JP5632610B2 - Optimized non-canonical zinc finger protein - Google Patents

Description

CROSS REFERENCE TO RELATED ART This application is incorporated by reference herein in its entirety, US Provisional Patent Application Nos. 60 / 874,911 and May 30, 2007, both filed December 14, 2006. Claims the benefit of the provisional US provisional application 60 / 932,497.

  The present disclosure belongs to the fields of genome engineering, gene targeting, target chromosomal integration, protein expression and epigenome editing.

  Sequence specific binding of proteins to DNA, RNA, proteins and other molecules is involved in many cellular processes such as transcription, replication, chromatin structure, recombination, DNA repair, RNA processing and translation. The binding specificity of cell-bound proteins involved in protein-DNA, protein-RNA and protein-protein interactions is involved in development, differentiation and homeostasis.

A zinc finger protein (ZFP) is a protein that can bind to DNA in a sequence-specific manner. Zinc fingers were first identified in the transcription factor TFIIIA from the oocyte of African clawed toad, Xenopus laevis. The single zinc finger domain of this class of ZFP is about 30 amino acids long, and some structural studies show that the domain contains a β-turn (containing two conserved cysteine residues) and an α-helix (two Which contain conserved histidine residues) and have been shown to be held in a specific conformation through coordination of the zinc atom by two cysteines and two histidines. This class of ZFP is also known as C ₂ H ₂ ZFP. A further class of ZFPs has also been suggested. See, for example, Jiang et al. (1996) J. Biol. Chem. 271: 10723-10730 for a discussion of Cys-Cys-His-Cys (C3H) ZFP. To date, over 10,000 zinc finger sequences have been identified in thousands of known or putative transcription factors. Zinc finger domains are involved not only in DNA recognition but also in RNA binding and protein-protein binding. Current estimates are that this class of molecules constitutes about 2% of all human genes.

Most zinc finger proteins have conserved cysteine and histidine residues coordinated tetrahedrally to one zinc atom in each finger domain. In particular, most ZFPs have the general sequence: -Cys- (X) _2-4 -Cys- (X) ₁₂ -His- (X) _3-5 -His- (SEQ ID NO: 1) [wherein X is Represents any amino acid] (C ₂ H ₂ ZFP) characterized by the finger component. This most widely represented class of zinc coordination sequences contains two cysteines and two histidines at specified intervals. Each finger's folded structure includes an antiparallel β-turn, a fingertip region, and a short amphiphilic α-helix. The ligand that coordinates to the metal binds to the zinc ion, and in the case of the zif268 type zinc finger, a short amphiphilic α-helix is bound to the main groove of DNA. In addition, the structure of zinc fingers was conserved and conserved by several conserved hydrophobic amino acid residues (eg, the residue immediately before the first conserved Cys and the +4 position residue of the finger helix segment) Stabilized by zinc coordination through cysteine and histidine residues.

Canonicals with changes in positions that directly contact the base, "supporting" or "buttressing" residues directly adjacent to the base contact position, and positions that can contact the phosphate backbone of DNA ( canonical) (C ₂ H ₂ ) zinc finger proteins have been described. For example, U.S. Patent Nos. 6,007,988; 6,013,453; 6,140,081; 6,866,997; 6,746,838; No. 6,140,081; No. 6,610,512; No. 7,101,972; No. 6,453,242; No. 6,785,613; No. 7,013,219 International Publication No. PCT WO 98/53059; Choo et al. (2000) Curr. Opin. Struct. Biol. 10: 411-416; Segal et al. (2000) Curr. Opin. Chem. Biol. 4:34 See -39.

  In addition, zinc finger proteins comprising zinc fingers with modified zinc coordination residues have also been described (eg, US Patent Application Nos. 20030108880, 20060246567, the disclosures of which are incorporated herein by reference. No. and No. 20060246588). However, zinc finger proteins, including these non-canonical zinc fingers, retain gene transcription regulatory functions, while the ability to act as zinc finger nucleases (ZFNs) is, in some cases, exclusively canonical. It is lower than the zinc finger protein composed of C2H2 zinc finger.

US Pat. No. 6,007,988 US Pat. No. 6,013,453 US Pat. No. 6,140,081 US Pat. No. 6,866,997 US Pat. No. 6,746,838 US Pat. No. 6,140,081 US Pat. No. 6,610,512 US Pat. No. 7,101,972 US Pat. No. 6,453,242 US Pat. No. 6,785,613 US Patent No. 7,013,219 International Publication No. PCT WO98 / 53059 US Patent Application No. 20030108880 US Patent Application No. 20060246567 US Patent Application No. 20060246588

Jiang et al. (1996) J. Biol. Chem. 271: 10723-10730 Choo et al. (2000) Curr. Opin. Struct. Biol. 10: 411-416; Segal et al. (2000) Curr. Opin. Chem. Biol. 4: 34-39

  Thus, there is still a need for further engineered zinc finger binding proteins, including zinc fingers with optimized non-canonical zinc coordination regions, particularly in the construction of zinc finger nucleases.

  The present disclosure provides a zinc finger DNA binding domain having changes in at least one zinc coordination residue. In particular, CCHC zinc fingers are now described. These CCHC zinc fingers have additional changes (substitutions) in the vicinity of the zinc coordinating residues, eg, the residues around the zinc coordinating residues that are closest to the C-terminal of the zinc finger (C-terminal-most). Insertions and / or deletions). Also disclosed are zinc finger polypeptides and fusion proteins comprising one or more of these CCHC zinc fingers, polynucleotides encoding these zinc fingers and fusion proteins, and methods of using these zinc finger polypeptides and / or fusion proteins. Is done.

Accordingly, the present disclosure includes, but is not limited to, the following numbered embodiments.
1. A zinc finger protein comprising a non-canonical (non-C ₂ H ₂ ) zinc finger, wherein the non-canonical zinc finger has a helical part involved in DNA binding, and the zinc coordination region of the helical part is an amino acid sequence HX ₁ The zinc finger protein comprising X ₂ RCX _L (SEQ ID NO: 2) and wherein the zinc finger protein is engineered to bind to a target sequence.
2. The zinc finger protein of embodiment 1, wherein X ₁ is A and X ₂ is Q.
3. The zinc finger protein of embodiment 1, wherein X ₁ is K and X ₂ is E.
4). The zinc finger protein of embodiment 1, wherein X ₁ is T and X ₂ is R.
5. X _L is G, the zinc finger protein of embodiment 1.
6). At least one zinc finger has the sequence Cys- (X ^A ) _2-4 -Cys- (X ^B ) ₁₂ -His- (X ^C ) _3-5 -Cys- (X ^D ) _1-10 (SEQ ID NO: 3) ^A zinc finger protein comprising two or more zinc fingers, including [wherein X ^A , X ^B , X ^C and X ^D may be any amino acid].
7). The zinc finger protein of any of embodiments 1-6, comprising any of the sequences set forth in any of Tables 1, 2, 3 or 4.
8). Either zinc finger protein that X ^D containing the sequence QLV or QKP, embodiment 6 or 7.
9. Embodiment 9. The zinc finger protein of embodiment 8, wherein the sequence QLV or QKP is the three C-terminal amino acid residues of the zinc finger.
10. X ^D comprises 1, 2 or 3 Gly (G) residues, or zinc finger protein of embodiment 6-9.
11. A zinc finger protein comprising a plurality of zinc fingers, wherein at least one of the zinc fingers comprises a CCHC zinc finger according to any of embodiments 1-10.
12 The zinc finger protein of embodiment 11, wherein the zinc finger protein comprises 3, 4, 5 or 6 zinc fingers.
13. The zinc finger protein of embodiment 11 or 12, wherein finger 2 comprises a CCHC zinc finger.
14 The zinc finger protein of any of embodiments 11-13, wherein the C-terminal zinc finger comprises a CCHC finger.
15. The zinc finger protein of any of embodiments 11-14, wherein the at least two zinc fingers comprise CCHC zinc fingers.
16. The zinc finger protein of any of embodiments 11-15, wherein the zinc finger protein comprises any of the sequences shown in Table 8 and is engineered to bind to a target sequence in the IPP2-K gene.
17. A fusion protein comprising any of the zinc finger proteins of embodiment 1-16 and one or more functional domains.
18. (A) a cut half domain,
(B) A fusion protein comprising the zinc finger protein of any of embodiments 1-16, and (c) a ZC linker interposed between the cleavage half domain and the zinc finger protein.
19. The fusion protein of embodiment 18, wherein the length of the ZC linker is 5 amino acids.
20. The fusion protein of embodiment 19, wherein the amino acid sequence of the ZC linker is GLRGS (SEQ ID NO: 4).
21. Embodiment 19. The fusion protein of embodiment 18, wherein the ZC linker is 6 amino acids in length.
22. The fusion protein of embodiment 21, wherein the amino acid sequence of the ZC linker is GGLRGS (SEQ ID NO: 5).
23. A polynucleotide encoding a zinc finger protein according to any of embodiments 1-16 or a fusion protein according to any of embodiments 17-22.
24. A method for targeted cleavage of cellular chromatin in a plant cell comprising expressing in a cell a pair of fusion proteins according to any of embodiments 18-22,
(A) the target sequence of the fusion protein is within 10 nucleotides of each other; and (b) the fusion protein dimerizes to cleave the DNA located between the target sequences.
Said method.
25. A method of targeted genetic recombination in a host plant cell, comprising:
(A) expressing a pair of fusion proteins according to any of embodiments 18-22 in a host cell, wherein the target sequence of the fusion protein is present at the selected host target locus, and (b) a host target Identifying a recombinant host cell that exhibits a sequence change at the locus.
26. 26. The method of any of embodiments 24 or 25, wherein the sequence change is a mutation selected from the group consisting of genetic material deletion, genetic material insertion, genetic material replacement, and any combination thereof.
27. 27. The method of any of embodiments 24-26, further comprising introducing an exogenous polynucleotide into the host cell.
28. Embodiment 28. The method of embodiment 27, wherein the exogenous polynucleotide comprises a sequence homologous to the host target locus.
29. 29. The method of any of embodiments 24-28, wherein the plant is selected from the group consisting of monocotyledonous plants, dicotyledonous plants, gymnosperms and eukaryotic algae.
30. 30. The method of embodiment 29, wherein the plant is selected from the group consisting of corn, rice, wheat, potato, soybean, tomato, tobacco, Brassicaceae and Arabidopsis.
31. The method of any of embodiments 24-29, wherein the plant is a tree.
32. 32. The method of any of embodiments 24-31, wherein the target sequence is in the IPP2K gene.
33. A method for reducing the level of phytic acid in seeds comprising inactivating or altering the IPP2-K gene according to embodiment 32.
34. A method for making phosphorus more metabolically usable in seeds, comprising inactivating or altering the IPP2-K gene according to embodiment 32.
35. A plant cell comprising a zinc finger protein according to any of embodiments 1-16 or a fusion protein according to any of embodiments 17-22 or a polynucleotide according to embodiment 23.
36. 36. The plant cell of embodiment 35, wherein the cell is a seed.
37. The plant cell of embodiment 36, wherein the seed is a corn seed.
38. The plant cell of any of embodiments 35-37, wherein IPP2-K is partially or fully inactivated.
39. 39. The plant cell of embodiment 38, wherein the level of phytic acid in the seed is reduced.
40. 40. The plant cell of embodiments 35-39, wherein the metabolically usable level of phosphorus in the cell is elevated.

FIG. 1 is a graph showing the rate of gene repair as measured by the percentage of cells expressing GFP in US 2005/0064474 and a GFP cell reporter assay system as described below. ZFN variants are represented by “XY”, where “X” refers to the table number and “Y” refers to the number given to the zinc finger in the specifically selected table. For example, “2-21” refers to a ZFN having a finger comprising the sequence shown in column 21 in Table 2, ie HAQRCGLRGSQLV (SEQ ID NO: 53). FIG. 2 is a graph showing the percentage of Cel-1 signal resulting from cleavage using various pairs of ZFN variants. The results of two experiments are shown for each pair of ZFNs with reference to the sample number. The mutant pairs used for each sample are shown in the box in the upper right corner, and “wt 5-8” and “wt 5-9” are shown in Example 14 (Table 5) of US Patent Application No. 2005/0064474. Refers to the canonical ZFN pair disclosed in 17). In sample 3-12, the C-terminal region of the recognition helix of finger 2 or finger 4 of canonical ZFN 5-8 or 5-9 is replaced with a non-canonical sequence. The partial sequence of non-canonical ZFN variants, designated 20, 21, 43, 45, 47 and 48 in sample 3-12 and the finger positions of these variants within the 4-finger ZFN are shown in the upper left of the graph. Shown in the corner. The asterisk above the bar showing the results from Experiment 2 for samples 8 and 9 indicates the background in the lane that causes an underestimation of ZFN efficiency. FIG. 3 is a graph showing gene repair rates in US 2005/0064474 and the GFP cell reporter assay system described herein. The ZFN pairs tested in each sample are shown below each bar, in which zinc finger numbers 20, 21, 43, 45, 47 and 48 are as described in Example 3, CCHC zinc fingers 1a- 10a contains the sequences shown in Tables 3 and 4. Zinc fingers 20, 21, 7a, 8a, 9a and 10a were used on finger 4; zinc fingers 43, 45, 47, 48, 1a, 2a, 3a, 4a, 5a and 6a were used on finger 2. FIG. 4 is a linear schematic representation of plasmid pDAB1585, a target vector for tobacco. FIG. 5 is a schematic representation of plasmid pDAB1585, a target vector for tobacco. Figures 6A and 6B show zinc finger nuclease (ZFN). FIG. 6A is a schematic representation of ZFN binding. FIG. 6B shows the sequence of the target sequence. FIG. 7 is a schematic representation of plasmid pDAB1400. FIG. 8 is a schematic representation of plasmid pDAB782. FIG. 9 is a schematic representation of plasmid pDAB1582. FIG. 10 is a schematic representation of plasmid pDAB354. FIG. 11 is a schematic representation of plasmid pDAB1583. FIG. 12 is a schematic representation of plasmid pDAB2407. FIG. 13 is a schematic representation of plasmid pDAB1584. FIG. 14 is a schematic representation of plasmid pDAB2418. FIG. 15 is a schematic representation of plasmid pDAB4045. FIG. 16 is a schematic representation of plasmid pDAB1575. FIG. 17 is a schematic representation of plasmid pDAB1577. FIG. 18 is a schematic representation of plasmid pDAB1579. FIG. 19 is a schematic representation of plasmid pDAB1580. FIG. 20 is a schematic representation of plasmid pDAB3401. FIG. 21 is a schematic representation of plasmid pDAB1570. FIG. 22 is a schematic representation of plasmid pDAB1572. FIG. 23 is a schematic representation of plasmid pDAB4003. FIG. 24 is a schematic representation of plasmid pDAB1571. FIG. 25 is a schematic representation of plasmid pDAB7204. FIG. 26 is a schematic representation of plasmid pDAB1573. FIG. 27 is a schematic representation of plasmid pDAB1574. FIG. 28 is a schematic representation of plasmid pDAB1581. FIG. 29 is a schematic representation of plasmid pDAB1576. FIG. 30 is a schematic representation of plasmid pDAB1600. FIG. 31 is a schematic representation of plasmid pDAB3731. FIG. 32 is a schematic representation of plasmid pDAB4322. FIG. 33 is a schematic representation of plasmid pDAB4331. FIG. 34 is a schematic representation of plasmid pDAB4332. FIG. 35 is a schematic representation of plasmid pDAB4333. FIG. 36 is a schematic representation of plasmid pDAB4334. FIG. 37 is a schematic representation of plasmid pDAB4336. Figure 38 is a schematic representation of plasmid pDAB4339. FIG. 39 is a schematic representation of plasmid pDAB4321. FIG. 40 is a schematic representation of plasmid pDAB4323. FIG. 41 is a schematic representation of plasmid pDAB4341. FIG. 42 is a schematic representation of plasmid pDAB4342. FIG. 43 is a schematic representation of plasmid pDAB4343. Figure 44 is a schematic representation of plasmid pDAB4344. FIG. 45 is a schematic representation of plasmid pDAB4346. FIG. 46 is a schematic representation of plasmid pDAB4330. FIG. 47 is a schematic representation of plasmid pDAB4351. FIG. 48 is a schematic representation of plasmid pDAB4356. FIG. 49 is a schematic representation of plasmid pDAB4359. FIG. 50 is a schematic representation of plasmid pDAB7002. FIG. 51 is a schematic representation of plasmid pDAB7025. FIG. 52 is a schematic representation of plasmid pDAB1591. FIG. 53 is a schematic representation of plasmid pcDNA3.1-SCD27a-L0-Fok1, which is a DNA template used for PCR amplification of Scd27 ZFN. FIG. 54 is a schematic representation of plasmid pDAB1594. FIG. 55 is a schematic representation of plasmid pDAB1598. FIG. 56 is a schematic representation of plasmid pDAB1577. FIG. 57 is a schematic representation of plasmid pDAB1578. FIG. 58 is a schematic representation of plasmid pDAB1601, a PAT gene control vector. FIG. 59 is a schematic representation of predicted intrachromosomal homologous recombination stimulated by IL-1-Fok1 fusion protein. FIG. 60 is a schematic representation of plasmid pDAB1590, a positive GFP expression control. FIG. 61 is a diagram showing predicted interchromosomal homologous recombination stimulated by IL-1 zinc finger-Fok1 fusion protein. FIG. 62 is a diagram showing predicted interchromosomal homologous recombination stimulated by Scd27 zinc finger-Fok1 fusion protein. FIG. 63 is a gel showing PCR analysis of recombinants. The first four lanes on the left are displayed at the top of the gel. Lanes labeled 1-5 show HR events from transformation of BY2-380 with C ₃ H IL-1-Fok1 fusion protein gene. Lanes labeled 6-7 show HR events from transformation of BY2-380 with the C ₃ H SCD27-Fok1 fusion protein gene. FIG. 64 shows the maize IPP2K gene sequence (SEQ ID NO: 6) derived from HiII cell culture used as a design template for ZFN engineering targeted to maize IPP2K. FIG. 65, panels AE show the ZFN expression vector cloning scheme. A stepwise cloning strategy was used to generate the ZFN expression construct. Individual ZFN-encoding genes were cloned into the vectors pVAX-N2A-NLSop2-EGFP-FokMono (A) and pVAX-C2A-NLSop2-EGFP-FokMono (B) to create a dual protein cassette (C) . This cassette was ligated to pDAB3872 (D) to make the final plasmid (E) for expression of the ZFN heterodimer. FIG. 66 shows ZFN binding in the maize IPP2K gene. Two ZFN proteins are required to perform double-strand breaks in DNA. The sequence around the cleavage site (indicated by the down arrow) is shown (SEQ ID NO: 7). One protein (8705) binds to the sequence CTGTGGGGCCAT (upper chain) (SEQ ID NO: 8) and the other protein (8684, 8585 or 8686) is the downstream sequence (CTTGACCACTACTCAGCCCAG, lower chain) (SEQ ID NO: 9 ). FIG. 67 shows the sequences of wild type (upper sequence, SEQ ID NO: 10) and ZFN clone 127 (lower sequence, SEQ ID NO: 11). The cleavage target for this ZFN is highlighted in a gray box. FIG. 68 shows an alignment of multiple deletions resulting from non-homologous end joining (NHEJ) of dsDNA breaks via ZFN within the maize IPP2K gene detected by 454 sequencing. The cleavage target for this ZFN is highlighted in a gray box. FIG. 69 is a graph showing the rate of gene repair in US 2005/0064474 and the GFP cell reporter assay system described herein. The ZFN pairs tested in each sample are shown below each bar. FIG. 70 shows plasmid pDAB7471 constructed as described in Example 18B. FIG. 71 shows plasmid pDAB7451 constructed as described in Example 18C. FIG. 72 is a diagram showing an exemplary autonomous herbicide resistance gene expression cassette. This construct comprises a complete promoter transcription unit (PTU) comprising a promoter, a herbicide resistance gene and a polyadenylation (polyA) termination sequence as described in Example 18D. FIG. 73 shows plasmid pDAB7422 constructed as described in Example 18E. This plasmid contains a complete promoter transcription unit (PTU) containing a promoter inserted in a position-1 plasmid backbone, a herbicide resistance gene and a polyadenylation (polyA) termination sequence. FIG. 74 shows plasmid pDAB7452 constructed as described in Example 18E. This plasmid contains a complete promoter transcription unit (PTU) containing a promoter, a herbicide resistance gene and a polyadenylation (polyA) termination sequence inserted into the position-2 plasmid backbone. FIG. 75 is a diagram showing an exemplary non-autonomous herbicide resistance gene expression cassette. This construct contains an incomplete promoter transcription unit (PTU) containing a herbicide resistance gene and a polyadenylation (polyA) termination sequence as described in Example 18F. FIG. 76 shows plasmid pDAB7423 constructed as described in Example 18G. This plasmid contains an incomplete promoter transcription unit (PTU) containing a herbicide resistance gene and a polyadenylation (polyA) termination sequence inserted into the position-1 plasmid backbone. FIG. 77 shows plasmid pDAB7454 constructed as described in Example 18G. This plasmid contains an incomplete promoter transcription unit (PTU) containing a herbicide resistance gene and a polyadenylation (polyA) termination sequence inserted into the position-2 plasmid backbone as described in Example 18G. FIG. 78 shows plasmid pDAB7424 (position-1 autonomous donor corresponding to exemplary Gateway®) constructed as described in Example 18H. FIG. 79 shows plasmid pDAB7425 (an exemplary Gateway®-compatible position-1 autonomous donor) constructed as described in Example 18H. FIG. 80 shows plasmid pDAB7426, constructed as described in Example 18H. pDAB7426 is a combination plasmid containing a ZFN expression cassette and a position-1 autonomous donor. FIG. 81 shows the plasmid pDAB7427 constructed as described in Example 18H. pDAB7427 is a combination plasmid containing a ZFN expression cassette and a position-1 autonomous donor. FIG. 82 shows amplification of donor DNA specific sequences from genomic DNA. The presence of the 317 bp product is indicated by the maize callus line no. Characteristic of the presence of donor DNA containing the PAT gene inserted into the 61-72 genome. HiII indicates a wild type negative control. FIG. 83 shows amplification of 5 'border sequences between donor DNA and maize genomic sequences specific for IPP2K. Secondary PCR products derived from the targeted integration of donors into the IPP2K gene were diagnosed by the presence of a 1.65 Kbp DNA fragment as described in Example 21A. HiII indicates a wild type negative control. FIG. 84 shows the amplification of the 3 'border sequence between the donor DNA and the maize genomic sequence specific for IPP2K. Secondary PCR products derived from the targeted integration of donors into the IPP2K gene were diagnosed by the presence of a 1.99 Kbp DNA fragment as described in Example 21A. HiII indicates a wild type negative control. Figure 85 shows amplification of the upstream (5 ') border sequence between the genome and the donor. PCR products derived from the targeted integration of donors into the IPP2K gene (5 'border sequence) were diagnosed by the presence of a 1.35 Kbp sized DNA fragment as described in Example 21B. HiII indicates a wild type negative control. FIG. 86 shows the amplification of the downstream (3 ′) border sequence between the donor and the genome. PCR products derived from the targeted integration of the donor into the IPP2K gene (3 'border sequence) were diagnosed by the presence of a 1.66 Kbp sized DNA fragment as described in Example 21B. HiII indicates a wild type negative control. FIG. 87 shows the sequence of the position-1 5′-homology flank (SEQ ID NO: 171). FIG. 88 shows the sequence of the position-1 3 ′ homology flank (SEQ ID NO: 172). FIG. 89 shows the sequence of the 5 ′ homology flank (SEQ ID NO: 139) at position-2. FIG. 90 shows the sequence of the position-2 3'-homology flank (SEQ ID NO: 140). Figure 91 shows the sequence of the (5 ') IPP2K genomic sequence (SEQ ID NO: 141) upstream of the ZFN target region. FIG. 92 shows the sequence of the (3 ′) IPP2K genomic sequence (SEQ ID NO: 142) downstream of the ZFN target region.

  Disclosed herein are compositions comprising a zinc finger binding polypeptide (ZFP) comprising non-canonical zinc fingers in a Cys-Cys-His-Cys pattern. Since zinc coordination provides the main folding energy for zinc fingers, the regulation of zinc coordination residues is responsible for various important functional features of zinc finger proteins such as cell half-life, other cellular factors It provides an easy means to modify the stability and structure of fingers that affect interactions, DNA binding specificity and affinity, and the relative orientation of functional domains.

  Zinc finger proteins, including those disclosed in non-canonical zinc fingers, such as those disclosed in US Patent Application Nos. 20030108880; 20060246567; and 200602246588, have been shown to bind to DNA and alter transcription. It was. However, when incorporated into zinc finger nucleases (ZFNs, see, eg, US Patent Application No. 2005/0064474), these previously described non-canonical zinc finger proteins sometimes cleave target DNA It may exhibit suboptimal activity.

  Described herein are zinc finger proteins comprising one or more CCHC zinc fingers in which specific sequences around the C-terminal pair of zinc coordination residues have been altered. Also, fusion proteins containing these optimized non-canonical zinc fingers whose ZFN cleaves the target DNA at a rate comparable to that achieved using ZFNs containing canonical (CCHH) zinc fingers For example, zinc finger nuclease (ZFN) is also described herein.

  A fusion polypeptide as disclosed herein can enhance or suppress gene transcription and / or cleave the target sequence. Polynucleotides encoding optimized non-canonical zinc fingers and polynucleotides encoding fusion proteins comprising one or more optimized non-canonical zinc fingers are also provided. In addition, therapeutically effective amounts of any of the zinc finger nucleotide binding polypeptides or functional fragments thereof described herein; or treatment of nucleotide sequences encoding any of the modified zinc finger nucleotide binding polypeptides or functional fragments thereof. Pharmaceutical compositions containing an effective amount in combination with a pharmaceutically acceptable carrier are provided. Further, an agriculturally effective amount of any of the zinc finger nucleotide binding polypeptides or functional fragments thereof described herein; or the agricultural science of nucleotide sequences encoding any of the modified zinc finger nucleotide binding polypeptides or functional fragments thereof. An agrochemical composition is provided that contains a pharmaceutically effective amount in combination with an agriculturally acceptable carrier. Also provided are screening methods for obtaining modified zinc finger nucleotide binding polypeptides that bind to genomic sequences.

  Genomic sequences include chromosomes, episomes, organelle genomes (eg mitochondria, chloroplasts), artificial chromosomes and any other type of nucleic acid present in the cell, such as amplified sequences, double microchromosomes and endogenous or infecting bacteria And those present in the viral genome. Genomic sequences can be normal (ie, wild type) or mutated; mutated sequences can include, for example, insertions, deletions, substitutions, translocations, rearrangements, and / or point mutations. The genomic sequence can also include one of many different alleles.

Overview The practice of the methods disclosed herein, as well as the preparation and use of the compositions disclosed herein, are within the technical scope of the art, unless otherwise indicated, and include molecular biology, biochemistry, chromatin structure structures and Conventional techniques in analysis, computer chemistry, cell culture, recombinant DNA and related fields are used. These techniques are explained in detail in the literature. For example, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates ; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, "Chromatin" (PM Wassarman and AP Wolffe, eds .), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, “Chromatin Protocols” (PB Becker, ed.) Humana Press, Totowa, 1999.

Definitions The terms “nucleic acid”, “polynucleotide” and “oligonucleotide” are used interchangeably to refer to a deoxyribonucleotide or ribonucleotide polymer in a linear or circular conformation, in single- or double-stranded form. Point to. For the purposes of this disclosure, these terms should not be construed as limitations on the length of the polymer. The term can encompass known analogs of natural nucleotides, as well as nucleotides that are modified in the base, sugar and / or phosphate moieties (eg, phosphorothioate backbone). In general, analogs of specific nucleotides have the same base-pairing specificity, ie, analogs of A base-pair with T.

  The terms “polypeptide”, “peptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues. These terms also apply to amino acid polymers in which one or more amino acids are chemical analogs or modified derivatives of the corresponding naturally occurring amino acids.

“Binding” refers to sequence-specific, non-covalent interactions between macromolecules (eg, between proteins and nucleic acids). As long as the interaction is overall sequence specific, not all components of the binding interaction need be sequence specific (eg, contact with phosphate residues within the DNA backbone). Such interactions are generally characterized by a dissociation constant (K _d ) of 10 ⁻⁶ M ⁻¹ or lower. “Affinity” refers to the strength of binding; high binding affinity correlates with lower K _d .

  A “binding protein” is a protein that can bind non-covalently to another molecule. The binding protein can bind to, for example, a DNA molecule (DNA binding protein), an RNA molecule (RNA binding protein) and / or a protein molecule (protein binding protein). In the case of protein binding proteins, they can bind to themselves (form homodimers, homotrimers, etc.) and / or bind to one or more molecules of one or more different proteins. Can do. A binding protein can have more than one type of binding activity. For example, zinc finger proteins have DNA binding, RNA binding and protein binding activities.

  A “zinc finger DNA binding protein” (or binding domain) is a sequence specific through one or more zinc fingers that is a region of the amino acid sequence within the binding domain whose structure is stabilized through the coordination of zinc ions. A protein that binds to DNA or a domain within a larger protein. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP.

  A zinc finger binding domain can be “engineered” to bind to a predetermined nucleotide sequence. Non-limiting examples of methods for manipulating zinc finger proteins are design and selection. A designed zinc finger protein is a non-naturally occurring protein whose design / composition arises primarily from rational criteria. Rational criteria for design include application of replacement rules and computer algorithms for processing information in databases that store information on existing ZFP designs and combined data. See, for example, U.S. Patent Nos. 6,140,081; 6,453,242; 6,534,261; and 6,785,613; and International Publication No. WO 98/53058. WO 98/53059; WO 98/53060; WO 02/016536; and WO 03/016496; and US Pat. Nos. 6,746,838; 6,866,997; See also 7,030,215.

  A “selected” zinc finger protein is a protein that is not found in nature, the production of which mainly results from empirical processes such as phage display, interaction traps or hybrid selection. For example, US Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,200,759; U.S. Pat. No. RE39,229; and International Publication No. WO95 / 19431; WO96 / 06166; WO98 / 53057; WO98 / 54311; WO00 / 27878. No .; WO01 / 60970; WO01 / 88197 and WO02 / 099084.

A “non-canonical” zinc finger protein is a protein that contains non-canonical (non-C2H2) zinc fingers. A non-canonical zinc finger thus comprises at least one amino acid substitution, addition and / or deletion compared to a naturally occurring C ₂ H ₂ zinc finger protein. Non-limiting examples of non-canonical zinc fingers are Cys-Cys-His-Cys zinc coordinating residues those containing (from amino terminus to carboxy terminus) (e.g. _C 3 H).

  “Homologous sequence” refers to a first sequence that shares some degree of sequence identity with a second sequence, which may be identical to the sequence of the second sequence. A “homologous non-identical sequence” refers to a first sequence that shares some sequence identity with a second sequence and whose sequence is not identical to the sequence of the second sequence. For example, a polynucleotide comprising a wild type sequence of a mutated gene is homologous and non-identical to the sequence of the mutated gene. In certain embodiments, the degree of homology between two sequences is sufficient to allow homologous recombination between them using normal cellular machinery. The two homologous non-identical sequences can be any length and the degree of non-homology can be as small as one nucleotide (eg, correction of genomic point mutations by targeted homologous recombination). Or 10 kilobases or larger (eg, for insertion of a gene at a predetermined site within a chromosome). Two polynucleotides comprising homologous non-identical sequences need not be the same length. For example, exogenous polynucleotides (ie donor polynucleotides) of 20-10,000 nucleotides or nucleotide pairs can be used.

Techniques for determining nucleic acid and amino acid sequence identity are known in the art. Typically, such techniques involve determining the nucleotide sequence of the mRNA for a gene and / or determining the amino acid sequence encoded thereby, and combining these sequences with the second nucleotide or amino acid sequence. Including comparing. Genomic sequences can also be determined and compared in this way. In general, identity refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their percent identity. The percent identity between two sequences is the number of exact matches between the two aligned sequences, divided by the length of the shorter sequence, multiplied by 100, regardless of the nucleic acid or amino acid sequence. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981). This algorithm was developed by Dayhoff, Atlas of Protein Sequences and Structure.MO Dayhoff ed., 5 suppl. 3: 353-358, National Biomedical Research Foundation, Washington, DC, USA.Gribskov, Nucl. Acids Res. 14 ( 6): 6745-6763 (1986) can be applied to amino acid sequences by using a scoring matrix normalized. An exemplary implementation of this algorithm for determining percent sequence identity is provided by the Genetics Computer Group (Madison, Wis.) In the “BestFit” utility application. Default parameters for this method are described in the Wisconsin Sequence Analysis Package Program Manual, Version 8 (1995) (available from Genetics Computer Group, Madison, Wis.). In the context of this disclosure, an exemplary method for establishing percent identity is described in John F. et al. Collins and Shane S. Developed by Sturrok, IntelliGenetics, Inc. (University View, CA) uses the MPSRCH package of a program owned by University of Edinburgh.
From this set of packages, the Smith-Waterman algorithm is available using default parameters for the scoring table (eg, 12 gap open penalty, 1 gap extension penalty, and 6 gaps). From the created data, the value of “Match” reflects “sequence identity”. Other suitable programs for calculating percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST used with default parameters. For example, BLASTN and BLASTP can be used with the following default parameters: Genetic Code = Standard; Filter = None; Chain = Both; Cutoff = 60; Expectation = 10; Matrix = BLOSUM62; Descriptions = 50 Sequences; Sort = HIGH SCORE; + Supdate + PIR. Details of these programs are detected on the Internet. For the sequences described herein, the desired extent of sequence identity is about 35% -100% and integer values in between. Typically, the percent identity between sequences is at least 35% -40%; 40% -45%; 45% -50%; 50% -60%; 60% -70%; 70-75%, preferably Is 80-82%, more preferably 85-90%, even more preferably 92%, even more preferably 95%, most preferably 98% sequence identity.

  Alternatively, the degree of sequence similarity between polynucleotides can be determined by hybridization of polynucleotides under conditions that allow the formation of stable duplexes between homologous regions, followed by single-strand specific nucleases. It can be determined by digestion and determination of the size of the digested fragment. Two nucleic acid or two polypeptide sequences, as determined using the methods described above, are those sequences that are at least about 70% -75%, preferably 80-82%, more preferably over the defined length of the molecule. Are substantially homologous to each other when they exhibit 85-90%, even more preferably 92%, even more preferably 95%, most preferably 98% sequence identity. As used herein, substantially homologous also refers to sequences that exhibit complete identity to a specified DNA or polypeptide sequence. DNA sequences that are substantially homologous can be identified in Southern hybridization experiments, eg, under stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, for example, Sambrook et al., Supra; Nucleic Acid Hybridization: A Practical Approach, editors B.D. Hames and S.J. Higgins, (1985) Oxford; Washington, DC; IRL Press.

  Selective hybridization of two nucleic acid fragments can be determined as follows. The degree of sequence identity between two nucleic acid molecules affects the efficiency and intensity of hybridization events between such molecules. A partially identical nucleic acid sequence will at least partially inhibit the hybridization of a completely identical sequence to a target molecule. Inhibition of hybridization of completely identical sequences can be accomplished by hybridization assays well known in the art (eg, Southern (DNA) blotting, Northern (RNA) blotting, solution hybridization, etc., Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, NY). Such assays can be performed using varying degrees of selectivity, for example using conditions ranging from low stringency to high stringency. When using low stringency conditions, a partial degree of sequence identity indicates that there is no non-specific binding so that the secondary probe will not hybridize to the target if there are no non-specific binding events. Secondary probes that are not even sex (eg, probes that have less than about 30% sequence identity with the target molecule) can be used to evaluate.

  When utilizing a hybridization-based detection system, a nucleic acid probe complementary to a reference nucleic acid sequence is selected, and then the probe and reference sequence selectively hybridize or bind to each other by selection of appropriate conditions. To form a double-stranded molecule. A nucleic acid molecule that can selectively hybridize to a reference sequence under moderately stringent hybridization conditions typically has at least about 70% sequence identity with the sequence of the selected nucleic acid probe. Hybridize under conditions that permit detection of a target nucleic acid sequence at least about 10-14 nucleotides in length. Stringent hybridization conditions typically permit detection of a target nucleic acid sequence at least about 10-14 nucleotides in length having greater than about 90-95% sequence identity with the sequence of a selected nucleic acid probe. . Useful hybridization conditions for probe / reference sequence hybridization in which the probe and reference sequence have a certain degree of sequence identity can be determined as known in the art (eg, Nucleic Acid Hybridization: A Practical Approach, editors BD Hames and SJ Higgins, (1985) Oxford; Washington, DC; see IRL Press).

  Conditions for hybridization are well known to those skilled in the art. Hybridization stringency refers to the extent to which hybridization conditions adversely affect the formation of hybrids containing mismatched nucleotides, and higher stringency correlates with lower tolerance for mismatched hybrids. Factors affecting the stringency of hybridization are well known to those skilled in the art and include, but are not limited to, temperature, pH, ionic strength, concentrations of organic solvents such as formamide and dimethyl sulfoxide. As known to those skilled in the art, hybridization stringency increases with higher temperatures, lower ionic strength and lower solvent concentrations.

With respect to stringency conditions for hybridization, it is well known in the art that a number of equivalent conditions can be used to establish a particular stringency, for example by changing the following factors. Sequence length and nature, base composition of various sequences, concentrations of salts and other hybridization solution components, presence or absence of blocking agents in the hybridization solution (eg, dextran sulfate and polyethylene glycol), hybridization reaction temperature and time Parameters as well as various cleaning conditions. Selection of a particular set of hybridization conditions is selected according to standard methods in the art (see, eg, Sambrook, et al., Molecular Cloning: A Laboratory Manual. Second Edition, (1989) Cold Spring Harbor, NY).

  “Recombination” refers to the process of exchange of genetic information between two polynucleotides. In the context of this disclosure, “homologous recombination (HR)” refers to a special form of such exchange that occurs, for example, during repair of double-strand breaks in cells. This process requires nucleotide sequence homology and uses a “donor” molecule that serves as a template for the repair of the “target” molecule (ie, the molecule that has undergone double-strand breaks), and the genetic information from the donor to the target. Are known variously, such as “non-crossover gene conversion” or “short tract gene conversion”. While not wishing to be bound by any particular theory, such a transition can be caused by mismatch repair of heteroduplex DNA formed between the cleaved target and the donor, and / or when the donor is one of the targets. It may include “synthesis-dependent chain annealing” and / or related processes used to resynthesize partial genetic information. Such specialized HR often causes a change in the sequence of the target molecule such that part or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.

  “Cleavage” refers to the destruction of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of phosphodiester bonds. Both single-strand breaks and double-strand breaks are possible, and double-strand breaks can occur as a result of two separate single-strand break events. DNA cleavage can result in the production of either blunt ends or sticky ends. In certain embodiments, a fusion polypeptide is used for target double-stranded DNA cleavage.

  A “cleavage domain” includes one or more polypeptide sequences that have catalytic activity for DNA cleavage. The cleavage domain can be included in a single polypeptide chain, or cleavage activity can result from the association of two (or more) polypeptides.

  A “cleavage half domain” is a polypeptide sequence that forms a complex with a second polypeptide (identical or different) that has cleavage activity (eg, double-strand cleavage activity).

  The terms “cleavage domain” and “cleavage half-domain” refer to wild-type domains and cleavage domains or cleavage half-domains that retain the ability to multimerize (eg, dimerize) to form a functional cleavage domain. Includes portions or mutants.

  “Chromatin” (chromatin) is a nucleoprotein structure that includes the cytoplasmic genome. Cellular chromatin contains nucleic acids, primarily DNA, and proteins including histone and non-histone chromosomal proteins. The majority of eukaryotic chromatin is present in the form of nucleosomes, the nucleosome core comprising about 150 base pairs of DNA linked to an octamer containing two of each of histones H2A, H2B, H3 and H4, and linker DNA (Of different lengths depending on the organism) extend between the nucleosome cores. One molecule of histone H1 generally binds to linker DNA. For the purposes of this disclosure, the term “chromatin” is intended to encompass all types of cellular nucleoprotein, both prokaryotic and eukaryotic. Cellular chromatin includes both chromosomal and episomal chromatin.

  A “chromosome” is a chromatin complex that contains all or part of the genome of a cell. A cell's genome is often characterized by its karyotype, which is a collection of all chromosomes that contain the cell's genome. The cell's genome may contain one or more chromosomes.

  An “episome” is a replicating nucleic acid, nucleoprotein complex, or other structure that includes nucleic acids that are not part of the chromosomal karyotype of a cell. Examples of episomes include plasmids and some viral genomes.

  An “accessible region” is a site in cellular chromatin where a target site present in a nucleic acid can be bound by an exogenous molecule that recognizes the target site. While not wishing to be bound by any particular theory, it is thought that accessible regions are regions that are not packaged within the nucleosome structure. The unambiguous structure of the accessible region can often be detected by its sensitivity to chemical and enzymatic probes such as nucleases.

  A “target site” or “target sequence” is a nucleic acid sequence that defines the portion of a nucleic acid to which a binding molecule binds provided that sufficient conditions for binding exist. For example, the sequence 5'-GAATTC-3 'is the target site for the EcoRI restriction endonuclease.

  An “exogenous” molecule is a molecule that is not normally present in a cell but can be introduced into a cell by one or more genetic, biochemical or other methods. “Normal presence in the cell” is determined with respect to the particular developmental stage and environmental conditions of the cell. Thus, for example, molecules that exist only during muscle embryogenesis are exogenous molecules with respect to adult muscle cells. Similarly, molecules that are induced by heat shock are exogenous molecules with respect to cells that are not heat shocked. An exogenous molecule can include, for example, a functional form of a dysfunctional endogenous molecule or a dysfunctional form of a normal functional endogenous molecule.

  Exogenous molecules include, among other things, small molecules, macromolecules generated by combinatorial chemical processes, such as proteins, nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, polysaccharides, some modified derivatives of the above molecules, or the above molecules Can be any complex containing one or more of: Nucleic acids include DNA and RNA, can be single stranded or double stranded, can be linear, branched or circular, and can be of any length. Nucleic acids include those capable of forming duplexes as well as triplex forming nucleic acids. See, for example, US Pat. Nos. 5,176,996 and 5,422,251. Proteins include DNA binding proteins, transcription factors, chromatin remodeling factors, methylated DNA binding proteins, polymerases, methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases and helicases However, it is not limited to these.

  The exogenous molecule can be the same type of molecule as the endogenous molecule, such as an exogenous protein or nucleic acid. For example, the exogenous nucleic acid may comprise an infecting viral genome, an Agrobacterium tumefacians T chain, a plasmid or episome introduced into the cell, or a chromosome not normally present in the cell. An exogenous nucleic acid or polynucleotide, however, can comprise a sequence that is homologous or identical to an endogenous sequence. With respect to a particular endogenous genomic region, “exogenous sequence” refers to a nucleotide sequence that is not present in that region. Such exogenous sequences may be present at another endogenous chromosomal location or not at all in the genome. Thus, exogenous polynucleotides can include both exogenous and endogenous sequences; for example, can include transgenes flanked by sequences homologous to a genomic region. Such exogenous nucleic acids are used in methods for targeted integration and targeted recombination as described below. Methods for introducing exogenous molecules into cells are known to those skilled in the art and include lipid-mediated transfer (ie, liposomes containing neutral and cationic lipids), electroporation, direct injection, cell fusion, particle guns, calcium phosphate co- Including but not limited to precipitation methods, DEAE dextran mediated transfer and viral vector mediated transfer.

  In contrast, “endogenous” molecules are those normally present in specific cells at specific developmental stages under specific environmental conditions. For example, endogenous nucleic acids can include chromosomes, mitochondria, chloroplast or other organelle genomes, or naturally occurring episomal nucleic acids. Additional endogenous molecules can include proteins such as transcription factors and enzymes.

  A “fusion” molecule is a molecule in which two or more subunit molecules are linked, eg, covalently. Subunit molecules can be molecules of the same chemical type or can be molecules of different chemical types. Examples of the first type of fusion molecule include, but are not limited to, a fusion protein (eg, a fusion between a ZFP DNA binding domain and a cleavage domain) and a fusion nucleic acid (eg, a nucleic acid encoding the fusion protein described above). . Examples of the second type of fusion molecule include, but are not limited to, a fusion between a triplex-forming nucleic acid and a polypeptide, and a fusion between a minor groove binder and a nucleic acid.

  Expression of the fusion protein in the cell can occur from delivery of the fusion protein to the cell or by delivery of the polynucleotide encoding the fusion protein to the cell, in which case the polynucleotide is transcribed and the transcript is translated. To produce a fusion protein. Trans-splicing, polypeptide cleavage and polypeptide ligation can also be involved in protein expression in cells. Methods for delivery of polynucleotides and polypeptides to cells are set forth elsewhere in this disclosure.

  In the context of this disclosure, a “gene” refers to the DNA region that encodes a gene product (see below) and the production of the gene product, regardless of whether such regulatory sequences are adjacent to the coding and / or transcriptional sequences. Includes all DNA regions to be regulated. Thus, a gene includes promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, border elements, origins of replication, matrix binding sites and locus control regions, but not necessarily It is not limited to these.

  “Gene expression” refers to the conversion of information contained in a gene into a gene product. A gene product can be a direct transcript of a gene (eg, mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or some other type of RNA) or a protein produced by translation of mRNA. Gene products are also RNA modified by processes such as capping, polyadenylation, methylation and editing, and for example methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation and glycosylation Proteins modified by

  “Modulation” of gene expression refers to a change in the activity of a gene. Regulation of expression can include, but is not limited to, gene activation and gene repression.

  “Plant” cells include, but are not limited to, monocotyledonous or dicotyledonous cells. Non-limiting examples of monocotyledons include cereal plants such as corn, rice, barley, oats, wheat, sorghum, rye, sugar cane, pineapple, onion, banana and coconut. Non-limiting examples of dicotyledonous plants include tobacco, tomato, sunflower, cotton, sugar beet, potato, lettuce, melon, soybean, canola (rapeseed) and alfalfa. Plant cells may be from any part of the plant and / or from any stage of plant development.

  A “region of interest” is a non-coding sequence within or adjacent to a gene that is desired to bind to any region of cellular chromatin, such as a gene or exogenous molecule. The binding can be for target DNA cleavage and / or target recombination. The region of interest can be present, for example, in the chromosome, episome, organelle genome (eg, mitochondria, chloroplast), or infecting viral genome. The region of interest can be in the coding region of a gene, in a transcribed non-coding region, such as in a leader sequence, trailer sequence or intron, or in a non-transcribed region upstream or downstream of the coding region. The region of interest may be as small as one nucleotide pair or up to 25,000 nucleotide pairs, or any integer number of nucleotide pairs.

  The terms “operably linked” and “operably linked” refer to two or more components (such as array elements), where both components function normally and at least one of the components is the other component. Used interchangeably with respect to the juxtaposition of two or more components that are arranged to be able to mediate a function exerted on at least one of the two. Illustratively, a transcriptional regulatory sequence, eg, a promoter, is operably linked to a coding sequence if the transcriptional regulatory sequence controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. ing. A transcriptional regulatory sequence is generally operably linked in cis with a coding sequence, but need not be directly adjacent to it. For example, an enhancer is a transcriptional regulatory sequence that is operably linked to a coding sequence, even if not adjacent to the coding sequence.

  With respect to fusion peptides, the term “operably linked” can refer to the fact that each of the components, even when not so linked, performs the same function as being linked to the other component. . For example, for a fusion polypeptide in which a ZFP DNA binding domain is fused to a cleavage domain, within the fusion polypeptide, a ZFP DNA binding domain portion can bind to its target site and / or its binding site, while the cleavage domain is A ZFP DNA binding domain and a cleavage domain are operably linked when the DNA can be cleaved in the vicinity of the target site.

  A “functional fragment” of a protein, polypeptide or nucleic acid is a protein, polypeptide or nucleic acid whose sequence is not identical to the full-length protein, polypeptide or nucleic acid but retains the same function as the full-length protein, polypeptide or nucleic acid. It is a nucleic acid. A functional fragment may have more, fewer or the same number of residues as the corresponding native molecule and / or may contain one or more amino acid or nucleotide substitutions. Methods for determining the function of a nucleic acid (eg, coding function, ability to hybridize to another nucleic acid) are well known in the art. Similarly, methods for determining protein function are also well known. For example, the DNA binding function of a polypeptide can be determined by, for example, a filter binding assay, an electrophoretic mobility shift assay, or an immunoprecipitation assay. DNA cleavage can be assayed by gel electrophoresis. See Ausubel et al., Supra. The ability of a protein to interact with another protein can be determined, for example, by co-immunoprecipitation, two-hybrid assays, or genetic and biochemical complementarity. See, for example, Fields et al. (1989) Nature 340: 245-246; US Pat. No. 5,585,245 and International Publication No. PCT WO 98/44350.

Zinc Finger Binding Domains Non-canonical zinc finger binding domains and polynucleotides encoding these zinc finger binding domains are described herein. In certain embodiments, the non-canonical zinc finger binding domain described herein is a C3H zinc finger in which one of the two conserved zinc coordinated histidine residues has been converted to cysteine. In an additional embodiment, the histidine residue closest to the C-terminus is converted to a cysteine residue to produce a “CCHC protein”.

A zinc finger binding domain can include one or more zinc fingers (eg, 2, 3, 4, 5, 6, 7, 8, 9 or more zinc fingers) and any target sequence (eg, genomic sequence). ) Can be manipulated. The zinc finger binding domain may bind to DNA, RNA and / or protein. Typically one zinc finger domain is about 30 amino acids long. Zinc fingers are canonical C ₂ H ₂ zinc fingers (ie zinc ions are coordinated by two cysteines and two histidine residues) and, for example, C ₃ H zinc fingers (three zinc ions) And non-canonical zinc fingers, which are coordinated by one cysteine residue and one histidine residue). See also U.S. Patent Application Nos. 20030108880, 20060246567, and 20060246588, the disclosures of which are incorporated herein by reference.

  Structural testing shows that canonical zinc finger domains (motifs) are held in two beta sheets (which are held in beta turns containing two invariant cysteine residues) and one alpha helix (two invariant histidine residues). ), And that they are held in a specific conformation through coordination of the zinc atom by two cysteines and two histidines. The non-canonical zinc fingers disclosed herein retain this β-β-α structure.

  The non-canonical zinc finger described herein can be a naturally occurring zinc finger binding domain. More typically, however, the non-canonical zinc finger described herein is one or more zinc, wherein at least one of the cysteine or histidine residues coordinated to zinc is replaced with one or more amino acids. Contains finger component. For example, in certain embodiments, the C-terminal His residue of the canonical zinc finger binding molecule is replaced with a Cys residue.

The CCHC zinc fingers described herein can also include one or more changes (relative to the sequence of naturally occurring C ₂ H ₂ zinc fingers) in the sequence of amino acid residues other than the zinc coordination residues. Such changes can include substitutions, deletions and / or insertions. The amino acid can vary anywhere within the zinc finger. Non-limiting examples of changes include: (1) substitution of one residue around the altered zinc coordination residue; (2) addition of extra residues before and after the altered zinc coordination residue (eg, His residue closest to the C-terminus) Is converted to a Cys residue, the addition of an extra amino acid residue may promote zinc coordination by compensating for a shorter cysteine side chain); and / or (3) a naturally occurring CCHC zinc Substitution of a residue located between the His and Cys residues of the finger to the corresponding region of a non-canonical CCHC zinc finger.

In certain embodiments, a zinc finger protein described herein has a helical portion in which a non-canonical zinc finger is involved in DNA binding, and the zinc coordination region of the helical portion is amino acid sequence HX ₁ X ₂ RCX _L (SEQ ID NO: 2 And at least one zinc finger including non-canonical (non-C ₂ H ₂ ) zinc fingers, wherein the zinc finger protein is engineered to bind to the target sequence. In some embodiments, X ₁ is A or K or T, X ₂ is Q or E or R, and X _L is G.

In other embodiments, non-canonical zinc fingers described herein have the general _{^{structure: Cys- (X A) 2-4 -Cys-}} (X B) 12 -His- (X C) 3-5 -Cys- ( X ^D ) _1-10 (SEQ ID NO: 3) [wherein X ^A , X ^B , X ^C and X ^D represent any amino acid]. In embodiments where X ^C comprises 3 residues, (i) at least one of these residues is altered relative to the canonical CCHC zinc finger; and / or (ii) X ^D is canonical. It contains at least one deletion, substitution or insertion compared to a CCHC zinc finger. In certain embodiments, ^{X D} comprises the sequence QLV or QKP. In other embodiments, ^XD comprises one or more (eg 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) Gly (G) residues.

The partial amino acid sequences of exemplary non-canonical zinc fingers (including the third zinc coordination residue and its C-terminal side) are shown in Tables 1, 2, 3 and 4. In all tables, the zinc coordinating residues (H and C) closest to the two C-termini (ie the third and fourth) are underlined. Changes (eg, substitutions, insertions, deletions) compared to the sequence of “wild-type” non-canonical fingers (second row of Tables 1 and 3) are indicated by double underlining.

  As described above, a ZFP can include any number of zinc finger binding domains, eg, at least three zinc fingers. Furthermore, one, two or more or all of the zinc fingers can be non-canonical zinc fingers as described herein.

  In certain embodiments, the finger closest to the C-terminus of a multi-finger zinc finger protein comprises a canonical zinc finger. In other embodiments, the finger closest to the C-terminus of a multi-finger zinc finger protein is one or more of the CCHC fingers described herein, eg, one or more of the zinc coordinating Cys residues closest to the C-terminus. Includes CCHC fingers containing amino acid insertions. See Examples 1-5, describing a 4-finger zinc finger protein, where finger 2 (F2) and / or finger 4 (F4) are non-canonical zinc fingers as described herein.

  The zinc finger binding domain can be engineered to bind to a selected sequence. For example, Beerli et al. (2002) Nature Biotechnol. 20: 135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70: 313-340; Isalan et al. (2001) Nature Biotechnol. 19: 656- 660; Segal et al. (2001) Curr. Opin. Biotechnol. 12: 632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10: 411-416. Engineered zinc finger binding domains may have novel binding specificities compared to naturally occurring zinc finger proteins. Methods of operation include, but are not limited to, rational design and various types of selection (eg, methods of screening multiple different zinc finger sequences against a single target nucleotide sequence). A rational design uses, for example, a database containing triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences to link each triplet or quadruplet nucleotide sequence to a particular triplet or quadruplet nucleotide sequence. Including associating with one or more amino acid sequences of zinc fingers. See, for example, commonly owned US Pat. Nos. 6,453,242 and 6,534,261. Additional design methods are disclosed, for example, in US Pat. Nos. 6,746,838; 6,785,613; 6,866,997; and 7,030,215. Enhancement of the binding specificity of zinc finger binding domains is described, for example, in co-owned US Pat. No. 6,794,136.

  Exemplary selection methods including phage display and two-hybrid systems are described in US Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453. No. 6,410,248; No. 6,140,466; No. 6,200,759; and No. 6,242,568; and International Publication No. WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and British Patent GB 2,338,237.

  Each zinc finger binds to a 3 nucleotide (ie triplet) sequence (or a 4 nucleotide sequence that can overlap by one nucleotide with the 4 nucleotide binding site of an adjacent zinc finger), so that the zinc finger binding domain is engineered to bind. The length of the sequence (eg, target sequence) determines the number of zinc fingers within the engineered zinc finger binding domain. For example, for ZFPs where the zinc finger motif does not bind to overlapping subsites, a 6 nucleotide target sequence is bound by a 2 finger binding domain; a 9 nucleotide target sequence is bound by a 3 finger binding domain, etc. The binding sites (ie, subsites) for individual zinc fingers in the target sequence need not be contiguous, and the length and nature of the amino acid sequence (ie, inter-finger linker) between the zinc fingers in the multi-finger binding domain Depending on it can be separated by one or several nucleotides. See, for example, US Pat. Nos. 6,479,626; 6,903,185 and 7,153,949 and US Patent Application No. 2003/0119023, the disclosures of which are incorporated herein by reference. .

  In a multi-finger zinc finger binding domain, adjacent zinc fingers can be separated by an amino acid linker sequence of about 5 amino acids (a so-called “canonical” inter-finger linker) or by one or more non-canonical linkers. See, for example, commonly owned US Pat. Nos. 6,453,242 and 6,534,261. For engineered zinc finger binding domains that contain four or more fingers, inserting a longer (“non-canonical”) inter-finger linker sequence between parts of the zinc fingers can result in binding affinity by binding domains and / or Or the specificity may be increased. See, for example, US Pat. No. 6,479,626 and US Patent Application No. 2003/0119023, the disclosures of which are incorporated herein by reference. Thus, multi-finger zinc finger binding domains can also be characterized with respect to the presence and location of non-canonical inter-finger linkers. The use of longer interfinger linkers can also facilitate the binding of zinc finger proteins to target sites containing non-adjacent nucleotides. As a result, one or more subsites within the target site for the zinc finger binding domain can be separated from each other by 1, 2, 3, 4, 5 or more nucleotides. As an example, a 4-finger binding domain may bind to a 13 nucleotide target site that includes two adjacent 3 nucleotide subsites, an intervening nucleotide, and two adjacent triplet subsites within the sequence.

  A target subsite is a nucleotide sequence (generally 3 or 4 nucleotides) joined by a single zinc finger. However, the target site need not be a multiple of 3 nucleotides. For example, when cross-strand interactions occur (see, eg, US Pat. Nos. 6,453,242 and 6,794,136) one or more of the individual zinc fingers of the multi-finger binding domain overlap. Can bind to quadruplet subsites. See also US Pat. Nos. 6,746,838 and 6,866,997. As an example, a three-finger binding domain can bind to a 10 nucleotide target site comprising three overlapping 4 nucleotide subsites.

  Selection of sequences within cellular chromatin (eg, target sites) for binding by zinc finger domains can be performed, for example, according to the methods disclosed in commonly owned US Pat. No. 6,453,242 (September 17, 2002). The patent also discloses methods for designing ZFPs that bind to selected sequences. It will be apparent to those skilled in the art that simple visual inspection of nucleotide sequences can also be used for target site selection. Thus, any means for target site selection can be used in the methods described herein.

  Multi-finger zinc finger proteins can be constructed by linking the resulting individual zinc fingers, eg, by design or selection. Alternatively, linking molecules consisting of two zinc fingers can be linked together using either canonical or longer non-canonical inter-finger linkers (see above) to generate 4 and 6 finger proteins. Can do. Such a two finger module can be obtained, for example, by selecting two adjacent fingers that bind to a specific 6 nucleotide target sequence for a multi-finger protein (generally 3 fingers). See, eg, International Publication No. WO 98/53057 and US Patent Application Publication No. 2003/0119023, the disclosures of which are incorporated herein by reference. Alternatively, a two-finger module can be constructed by assembly of individual zinc fingers.

  Thus, the zinc finger domains described herein can be used individually or in various combinations to construct multi-finger zinc finger proteins that bind to some target site.

  The distance between sequences (eg, target sites) refers to the number of nucleotides or nucleotide pairs intervening between the two sequences as measured from the ends of the sequences closest to each other.

  For example, in embodiments using ZFN where cleavage is dependent on the binding of two zinc finger domain / cleaving half-domain fusion molecules to separate target sites, the two target sites may be on opposite DNA strands. In other embodiments, both target sites are on the same DNA strand. See, eg, International Publication No. WO 2005/084190, the disclosure of which is incorporated herein by reference.

  Polynucleotides that encode zinc fingers or zinc finger proteins are also within the scope of this disclosure. These polynucleotides can be constructed and inserted into the vector using standard techniques, and the vector can be introduced into the cell such that the encoded protein is expressed in the cell (polynucleotides into the cell). (See below for further disclosure regarding vectors and methods for introduction into).

Fusion proteins Fusion proteins comprising one or more non-canonical zinc finger components as described herein are also provided.

  Fusion molecules are constructed by cloning and biochemical coupling methods well known to those skilled in the art. Fusion molecules include CCHC-containing ZFPs and, for example, cleavage domains, cleavage half-domains, transcription activation domains, transcription repression domains, components of chromatin remodeling complexes, insulator domains, functional fragments of any of these domains; and And / or some combination of two or more functional domains or fragments thereof.

  In certain embodiments, the fusion molecule comprises a modified plant zinc finger protein and at least two functional domains (eg, an insulator domain or a methyl binding protein domain, and further a transcriptional activation or repression domain).

  The fusion molecule also optionally includes a nuclear localization signal (eg, from SV40 T antigen or maize Opaque-2 NLS) and an epitope tag (eg, FLAG or hemagglutinin). Fusion proteins (and the nucleic acids that encode them) are designed so that translational reading frames are conserved among the components of the fusion.

  Methods for the design and construction of fusion proteins (and the polynucleotides encoding them) are known to those skilled in the art. Methods for the design and construction of fusion proteins, including, for example, zinc finger proteins (and polynucleotides encoding them) are described in commonly owned US Pat. Nos. 6,453,242 and 6,534,261. Yes.

  Polynucleotides encoding such fusion proteins are also within the scope of this disclosure. These polynucleotides can be constructed and inserted into vectors using standard techniques, and the vectors can be introduced into cells (further disclosure regarding vectors and methods for introducing polynucleotides into cells). See below for details).

  An exemplary functional domain for fusing with the ZFP DNA binding domain used to suppress gene expression is the KRAB repression domain from human KOX-1 protein (eg Thiesen et al., New Biologist 2 , 363-374 (1990); Margolin et al., Proc. Natl. Acad. Sci. USA 91, 4509-4513 (1994); Pengue et al., Nucl. Acids Res. 22: 2908-2914 (1994); Witzgall et al., Proc. Natl. Acad. Sci. USA 91, 4514-4518 (1994)). The KOX domain is also suitable for use as a repression domain. Another suitable repression domain is methyl binding domain protein 2B (MBD-2B) (see also Hendrich et al. (1999) Mamm Genome 10: 906-912 for description of MBD proteins). Another useful repression domain is one that is associated with the v-ErbA protein. For example, Damm, et al. (1989) Nature 339: 593-597; Evans (1989) Int. J. Cancer Suppl. 4: 26-28; Pain et al. (1990) New Biol. 2: 284-294; Sap et al. (1989) Nature 340: 242-244; Zenke et al. (1988) Cell 52: 107-119; and Zenke et al. (1990) Cell 61: 1035-1049. Further exemplary repression domains include thyroid hormone receptor (TR), SID, MBD1, MBD2, MBD3, MBD4, MBD-like proteins, members of the DNMT family (eg, DNMT1, DNMT3A, DNMT3B), Rb, MeCP1 and MeCP2. However, it is not limited to these. For example, Zhang et al. (2000) Ann Rev Physiol 62: 439-466; Bird et al. (1999) Cell 99: 451-454; Tyler et al. (1999) Cell 99: 443-446; Knoepfler et al. 1999) Cell 99: 447-450; and Robertson et al. (2000) Nature Genet. 25: 338-342. Further exemplary repression domains include but are not limited to ROM2 and AtHD2A. See, for example, Chern et al. (1996) Plant Cell 8: 305-321; and Wu et al. (2000) Plant J. 22: 19-27.

  Suitable domains for achieving activation include HSV VP16 activation domains (see, eg, Hagmann et al., J. Virol. 71, 5952-5962 (1997)), nuclear hormone receptors (eg, Torchia et al. , Curr. Opin. Cell. Biol. 10: 373-383 (1998)); p65 subunit of nuclear factor κB (Bitko & Barik, J. Virol. 72: 5610-5618 (1998) and Doyle & Hunt, Neuroreport 8: 2937-2942 (1997)); Liu et al., Cancer Gene Ther. 5: 3-28 (1998)), or artificial chimeric functional domains such as VP64 (Seifpal et al., EMBO J. 11, 4961-4968 (1992)).

  Further exemplary activation domains include, but are not limited to, p300, CBP, PCAF, SRC1 PvALF and ERF-2. For example, Robyr et al. (2000) Mol. Endocrinol. 14: 329-347; Collingwood et al. (1999) J. Mol. Endocrinol. 23: 255-275; Leo et al. (2000) Gene 245: 1-11 ; Manteuffel-Cymborowska (1999) Acta Biochim. Pol. 46: 77-89; McKenna et al. (1999) J. Steroid Biochem. Mol. Biol. 69: 3-12; Malik et al. (2000) Trends Biochem. Sci. 25: 277-283; and Lemon et al. (1999) Curr. Opin. Genet. Dev. 9: 499-504. Further exemplary activation domains include, but are not limited to, OsGAI, HALF-1, C1, AP1, ARF-5, -6, -7 and -8, CPRF1, CPRF4, MYC-RP / GP and TRAB1. Not. For example, Ogawa et al. (2000) Gene 245: 21-29; Okanami et al. (1996) Genes Cells 1: 87-99; Goff et al. (1991) Genes Dev. 5: 298-309; Cho et al. (1999) Plant Mol. Biol. 40: 419-429; Ulmason et al. (1999) Proc. Natl. Acad. Sci. USA 96: 5844-5849; Sprenger-Haussels et al. (2000) Plant J. 22: 1-8; Gong et al. (1999) Plant Mol. Biol. 41: 33-44; and Hobo et al. (1999) Proc. Natl. Acad. Sci. USA 96: 15,348-15,353.

  Additional functional domains are disclosed, for example, in commonly owned US Pat. No. 6,933,113. In addition, insulator domains, chromatin remodeling proteins, such as ISWI-containing domains, and methyl-binding domain proteins suitable for use in fusion molecules are described, for example, in co-published international publications WO 01/83793 and WO 02/26960. ing.

  In other embodiments, the fusion protein is a zinc finger nuclease (ZFN) comprising one or more CCHC zinc fingers described herein and a cleavage domain (or cleavage half-domain). A zinc finger can be engineered to recognize a target sequence in any selected genomic region and, when introduced into a cell, binds the fusion protein to its binding site and in or near the genomic region. Cause cutting. Such truncation can cause changes in the nucleotide sequence of the genomic region (eg, mutation) after non-homologous end joining. Alternatively, if exogenous polynucleotides containing sequences homologous to the genomic region are simultaneously present in such cells, homologous recombination can occur between the genomic region and the exogenous polynucleotide at a high rate after target cleavage by ZFN. Occur. Homologous recombination can result in target sequence substitution or targeted integration of the exogenous sequence, depending on the nucleotide sequence of the exogenous polynucleotide.

  The non-canonical zinc fingers described herein provide improved cutting function when incorporated into ZFNs. As described in the Examples, a 4-finger ZFN containing at least one CCHC finger as described herein cleaves to the same extent as a nuclease containing at least exclusively CCHH fingers. In certain embodiments, when the C-terminal finger comprises a non-canonical CCHC zinc finger, the residues between the third and fourth zinc coordination residues (ie, between the C-terminal His and Cys residues) are Unlike those present in canonical CCHH zinc fingers, one or more glycine residues (eg, 1, 2, 3, 4, 5 or more) are inserted after the C-terminal Cys residue.

  The cleavage domain portion of ZFN disclosed herein can be obtained from any endonuclease or exonuclease. Exemplary endonucleases from which the cleavage domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, 2002-2003 Catalog, New England Biolabs, Beverly, MA; and Belfort et al. (1997) Nucleic Acids Res. 25: 3379-3388. Additional enzymes that cleave DNA are known (eg, S1 nuclease; mung bean nuclease; pancreatic DNase I; staphylococcal nuclease; yeast HO endonuclease; Linn et al. (Eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993 See also). One or more of these enzymes (or functional fragments thereof) can be used as a source of cleavage domains and cleavage half domains.

  Similarly, the cleavage half domain may be derived from any nuclease or portion thereof as indicated above, provided that the cleavage half domain requires dimerization for cleavage activity. In general, if the fusion protein contains a cleavage half domain, two fusion proteins are required for targeted cleavage of genomic DNA. Alternatively, a single protein containing two truncated half domains can be used. The two cleaved half domains can be derived from the same endonuclease, or each cleaved half domain can be derived from a different endonuclease. In addition, the target sites for the two fusion proteins can be linked to each other allowing the binding of the two fusion proteins to the respective target sites to form a functional cleavage domain, for example by dimerization. Positioned relative to each other so as to position the cut half domains in a spatial direction relative to each other. Thus, in certain embodiments, the proximal ends of the target sites are separated by 5-8 nucleotide pairs or 15-18 nucleotide pairs. In additional embodiments, the target sites are within 10 nucleotide pairs of each other. However, any integer number of nucleotides or nucleotide pairs can intervene between two target sites (eg 2-50 nucleotides or more). In general, the breakpoint is located between the target sites.

  Restriction endonucleases (restriction enzymes) are present in many species and can bind sequence-specifically to DNA (of the recognition site) and can cleave DNA at or near the binding site. Certain restriction enzymes (eg, type IIS) cleave DNA at sites away from the recognition site and have separable binding and cleavage domains. For example, the type IIS enzyme Fok I catalyzes double-strand breaks in DNA at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other strand. For example, US Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; and Li et al. (1992) Proc. Natl. Acad. Sci. USA 89: 4275- 4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90: 2764-2768; Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91: 883-887; Kim et al. (1994b) See J. Biol. Chem. 269: 31,978-31,982. Thus, in one embodiment, the fusion protein comprises a cleavage domain (or cleavage half-domain) from at least one type IIS restriction enzyme and one or more that may or may not be engineered. Of zinc finger binding domains.

  An exemplary type IIS restriction enzyme whose cleavage domain is separable from the binding domain is Fok I. This special enzyme is active as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10,570-10,575. Thus, for the purposes of this disclosure, the portion of the Fok I enzyme used in the disclosed fusion protein is considered a cleavage half domain. Thus, for targeted double-strand breaks using ZFNs containing zinc finger-Fok I fusions and / or targeted replacement of cellular sequences, two fusion proteins, each containing a Fok I cleavage half-domain, are catalytically cleaved. Can be used to reconfigure the domain. Alternatively, a single polypeptide molecule comprising one zinc finger binding domain and two Fok I cleavage half domains can be used. Parameters for target cleavage and target sequence alteration using zinc finger-Fok I fusions are described elsewhere in this disclosure and, for example, U.S. Patent Application No. Provided in 2005/0064474.

  In an additional embodiment, the FokI cleavage half domain may contain one or more mutations in any amino acid residue that affects dimer formation. Such mutations can be useful to prevent one of the ZFP / FokI fusion pairs from undergoing homodimerization that can lead to cleavage at undesired sequences. For example, the amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537 and 538 of FokI all affect dimerization. Is close enough to the dimer formation interface. Thus, amino acid sequence changes at one or more of the above positions can be used to change the dimerization properties of the truncated half domain. Such changes can be made, for example, by building a library containing (or encoding) different amino acid residues at these positions and selecting mutants with the desired properties, or rationalizing individual mutants It can be introduced by designing. In addition to preventing homodimerization, some of these mutations can also increase cleavage efficiency compared to those obtained with two wild-type cleavage half-domains.

  Thus, for targeted cleavage using a pair of ZFP / FokI fusions, one or both of the fusion proteins inhibits self-dimerization, but the two fusion proteins so that cleavage occurs at the desired target site. One or more amino acid changes that may result in heterodimerization of In certain embodiments, the change is present in both fusion proteins and the change has an additional effect, i.e., homodimerization of any fusion that leads to aberrant cleavage is minimized or It is eliminated, while heterodimer formation of the two fusion proteins is promoted compared to that obtained with the wild-type cleavage half-domain.

  In certain embodiments, the cleavage domain comprises two cleavage half domains, a first cleavage half domain and a second cleavage half domain, both of which are part of a single polypeptide comprising a binding domain. The cleaved half domains can have the same amino acid sequence or different amino acid sequences as long as they function to cleave DNA.

  The cleaved half domain can also be provided in a separate molecule. For example, two fusion polypeptides can be expressed in one cell, each polypeptide comprising a binding domain and a cleavage half domain. The cleaved half domains can have the same amino acid sequence or different amino acid sequences as long as they function to cleave DNA. Furthermore, the binding domain typically allows two cleavage half domains to reconstitute the cleavage domain (eg, by dimerization of the half domains) when bound to a fusion polypeptide. Bound to target sequences that are presented in a spatial orientation so that the half domains are positioned relative to each other to form a functional cleavage domain and cause cleavage of cellular chromatin within the region of interest To do. In general, cleavage by a reconstituted cleavage domain occurs at a site located between two target sequences. One or both of the proteins can be engineered to bind to its target site.

  Expression of two fusion proteins in a cell is the delivery of two proteins into the cell; the delivery of one protein and one nucleic acid encoding one of the proteins into the cell; each one of the proteins Can result from the delivery of two nucleic acids encoding to a cell; or the delivery of a single nucleic acid encoding both proteins to a cell. In an additional embodiment, the fusion protein comprises a single polypeptide chain comprising two cleavage half domains and one zinc finger binding domain. In this case, one fusion protein is expressed in the cell and, although not wishing to be bound by theory, is thought to cleave DNA as a result of the formation of an intramolecular dimer of the cleaved half domain.

  In certain embodiments, the components of ZFN are positioned such that the zinc finger domain is closest to the amino terminus of the fusion protein and the cleavage half domain is closest to the carboxy terminus. This demonstrates the relative orientation of the cleavage domain in naturally occurring dimerization cleavage domains, such as those derived from the Fok I enzyme where the DNA binding domain is closest to the amino terminus and the cleavage half domain is closest to the carboxy terminus. reflect. In these embodiments, dimerization of the cleaved half-domain to form a functional nuclease binds the fusion protein to a site on the opposite DNA strand and the 5 ′ ends of the binding sites are proximal to each other Caused by that.

  In this orientation, the zinc finger closest to the C-terminus is proximal to the FokI cleavage half domain. It was previously determined that non-canonical zinc finger proteins bind to their DNA targets most efficiently when CCHC-type zinc fingers are present as the fingers closest to the C-terminus. Therefore, the presence of a CCHC type zinc finger close to the FokI cleavage half domain described above may have inhibited its function. If so, the optimized CCHC zinc finger disclosed herein apparently does not show this hypothetical inhibitory activity.

  In additional embodiments, the components of the fusion protein (eg, a ZFP-Fok I fusion) are positioned so that the cleavage half domain is closest to the amino terminus of the fusion protein and the zinc finger domain is closest to the carboxy terminus. In these embodiments, dimerization of the cleaved half domain to form a functional nuclease binds the fusion protein to a site on the opposite DNA strand and the binding site 3 'ends are proximal to each other Caused by that.

  In a further additional embodiment, the first fusion protein comprises a cleavage half domain closest to the amino terminus of the fusion protein and a zinc finger domain closest to the carboxy terminus, and the second fusion protein is fused to the zinc finger domain. The protein is positioned closest to the amino terminus and the cleavage half domain closest to the carboxy terminus. In these embodiments, both fusion proteins bind to the same DNA strand, and the binding site of the first fusion protein containing the zinc finger domain closest to the carboxy terminus is the second containing the zinc finger domain closest to the amino terminus. It is located 5 'to the binding site of the fusion protein. See also International Publication No. WO2005 / 084190, the disclosure of which is incorporated herein by reference.

  The amino acid sequence between the zinc finger domain and the cleavage domain (or cleavage half domain) is referred to as a “ZC linker”. ZC linkers should be distinguished from the inter-finger linkers discussed above. For details regarding obtaining ZC linkers that optimize cleavage, see, for example, US Patent Publication Nos. 20050064474A1 and 20030232410, and International Publication No. WO 2005/084190, the disclosures of which are incorporated herein by reference. reference.

Expression vectors Nucleic acids encoding one or more ZFPs or ZFP fusion proteins (eg, ZFN) can be cloned into vectors for transformation into prokaryotic or eukaryotic cells for replication and / or expression. The vector can be a prokaryotic or eukaryotic vector, such as a plasmid, shuttle vector, insect vector, binary vector (eg, US Pat. No. 4,940,838; Horsch et al (1984) Science 233: 496-498, and Fraley et al (1983) Proc. Nat'l. Acad. Sci. USA 80: 4803) and the like. Nucleic acids encoding ZFPs can also be cloned into expression vectors for administration to plant cells.

  In order to express the fusion protein, the sequence encoding the ZFP or ZFP fusion is typically subcloned into an expression vector containing a promoter that directs transcription. Suitable bacterial and eukaryotic promoters are well known in the art, e.g. Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989; 3rd ed., 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al, supra). Bacterial expression systems for expressing ZFP are available, for example, in E. coli, Bacillus sp. And Salmonella (Palva et al, Gene 22: 229-235 (1983)). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast and insect cells are well known to those skilled in the art and are also commercially available.

  The promoter used to direct the expression of the nucleic acid encoding ZFP will depend on the particular application. For example, a strong constitutive promoter compatible with the host cell is typically used for the expression and purification of ZFP.

  In contrast, when ZFP is administered in vivo for plant gene regulation (see the section "Nucleic acid delivery to plant cells" below), a constitutive or inducible promoter depends on the specific use of ZFP. used. Non-limiting examples of plant promoters include Arabidopsis A. thaliana ubiquitin-3 (ubi-3) (Callis, et al., 1990, J. Biol. Chem. 265-12486-12493); A. tumefaciens Mannopine synthase (Δmas) (Petolino et al., US Pat. No. 6,730,824); and / or cassava vein mosaic virus (CsVMV) (Verdaguer et al., 1996, Plant Molecular Biology 31: 1129- 1139). See also Examples.

  In addition to the promoter, the expression vector typically includes a transcription unit or expression cassette that contains all the additional elements required for the expression of the nucleic acid in either prokaryotic or eukaryotic host cells. A typical expression cassette is thus required, for example, for a promoter operably linked to a nucleic acid sequence encoding ZFP and for efficient polyadenylation, transcription termination, ribosome binding site or translation termination of the transcript, for example. Contains signal. Additional elements of the cassette can include, for example, enhancers and heterologous splicing signals.

  The specific expression vector used to transport the genetic information into the cell is selected for the intended use of the ZFP, eg for expression in plants, animals, bacteria, fungi, protozoa, etc. (see expression vectors described below) . Standard bacterial and animal expression vectors are known in the art, eg, US Patent Publication No. 20050064474A1 and International Publication Nos. WO05 / 084190, WO05 / 90, the disclosures of which are incorporated herein by reference. No. 014791 and WO 03/080809 are described in detail.

  Standard transfection methods can be used to produce bacterial, mammalian, yeast or insect cell lines that express large amounts of protein, which can then be purified using standard techniques (eg, Collley et al. , J. Biol. Chem. 264: 17619-17622 (1989); Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells is performed according to standard techniques (eg Morrison, J. Bact. 132: 349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology 101: 347-362 (Wu et al, eds., 1983)).

  Any of the well-known procedures for introducing heterologous nucleotide sequences into such host cells can be used. These include calcium phosphate transfection, polybrene, protoplast fusion, electroporation, ultrasound (eg, sonoporation), liposomes, microinjection, naked DNA, plasmid vectors, both episomal and integrative viral vectors, and cloning Including the use of any of the other well-known methods for introducing engineered genomic DNA, cDNA, synthetic DNA or other heterologous genetic material into a host cell (see, eg, Sambrook et al, supra). The particular genetic engineering procedure used is sufficient if at least one gene can be successfully introduced into a host cell capable of expressing the selected protein.

Nucleic Acid Delivery to Plant Cells As noted above, DNA constructs can be introduced into a desired plant host (eg, into its genome) by a variety of conventional techniques. For a review of such methods, see, for example, Weissbach & Weissbach Methods for Plant Molecular Biology (1988, Academic Press, NY) Section VIII, pp. 421-463; and Grierson & Corey, Plant Molecular Biology (1988, 2d Ed.) , Blackie, London, Ch. 7-9.

  For example, the DNA construct can be introduced into plant cells using techniques such as electroporation and microinjection of plant cell protoplasts, or the DNA construct can be directly applied to plant tissue using particle bombardment, such as DNA particle bombardment. (See, for example, Klein et al (1987) Nature 327: 70-73). Alternatively, the DNA construct can be combined with an appropriate T-DNA flanking region and introduced into a conventional Agrobacterium tumefaciens host vector. Agrobacterium tumefaciens-mediated transformation techniques, including disarming and the use of binary vectors, are well described in the academic literature. See, for example, Horsch et al (1984) Science 233: 496-498, and Fraley et al (1983) Proc. Nat'l Acad. Sci. USA 80: 4803.

  In addition, gene transfer may include non-Agrobacterium bacteria or viruses such as Rhizobium NGR234, Sinorhizoboium meliloti, Mesorhizobium loti, potato X virus, cauliflower mosaic virus and cassava vein mosaic virus and / or Or it can be achieved using tobacco mosaic virus. See, for example, Chung et al. (2006) Trends Plant Sci. 11 (1): l-4.

  The virulence function of the Agrobacterium tumefaciens host is determined by using a binary T DNA vector (Bevan (1984) Nuc. Acid Res. 12: 8711-8721) or a co-culture procedure (Horsch et al (1985) Science 227: 1229-1231). When used to infect cells with bacteria, direct the insertion of the construct and adjacent markers into the plant cell DNA. In general, the Agrobacterium transformation system is used to manipulate dicotyledons (Bevan et al (1982) Ann. Rev. Genet 16: 357-384; Rogers et al (1986) Methods Enzymol. 118: 627 -641). The Agrobacterium transformation system can also be used to transform monocotyledonous plants and plant cells and to introduce DNA into them. U.S. Pat.No. 5,591,616; Hernalsteen et al (1984) EMBO J 3: 3039-3041; Hooykass-Van Slogteren et al (1984) Nature 311: 763-764; Grimsley et al (1987) Nature 325: 1677 -179; Boulton et al (1989) Plant Mol. Biol. 12: 31-40 .; and Gould et al (1991) Plant Physiol. 95: 426-434.

  Selective gene transfer and transformation methods include protoplast transformation through the incorporation of naked DNA via calcium, polyethylene glycol (PEG) or electroporation (Paszkowski et al. (1984) EMBOJ 3: 2717-2722, Potrykus et al. (1985) Molec. Gen. Genet. 199: 169-177; Fromm et al. (1985) Proc. Nat. Acad. Sci. USA 82: 5824-5828; and Shimamoto (1989) Nature 338: 274-276 Reference) and electroporation of plant tissue (D'Halluin et al. (1992) Plant Cell 4: 1495-1505), but is not limited thereto. Additional methods for plant cell transformation include microinjection, DNA incorporation via silicon carbide (Kaeppler et al. (1990) Plant Cell Reporter 9: 415-418), and microprojectile bombardment (Klein et al. (1988) Proc. Nat. Acad. Sci. USA 85: 4305-4309; and Gordon-Kamm et al. (1990) Plant Cell 2: 603-618).

  The disclosed methods and compositions can be used to insert exogenous sequences at predetermined locations within the plant cell genome. This is useful because the expression of the transgene introduced into the plant genome is critically dependent on its integration site. Thus, for example, genes encoding nutrients, antibiotics or therapeutic molecules can be inserted by targeted recombination into regions of the plant genome that favor their expression.

  Transformed plant cells produced by any of the above transformation techniques have a transformed genotype and can therefore be cultured to regenerate whole plants having the desired phenotype. Such regeneration techniques are based on manipulation of certain plant hormones in tissue culture growth media, typically based on biocide and / or herbicide markers introduced with the desired nucleotide sequence. Plant regeneration from cultured protoplasts is described in Evans, et al., `` Protoplasts Isolation and Culture '' in Handbook of Plant Cell Culture, pp. 124-176, Macmillian Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, organs, pollen, embryos or parts thereof. Such regeneration techniques are generally described in Klee et al (1987) Ann. Rev. of Plant Phys. 38: 467-486.

  Nucleic acids introduced into plant cells can be used to confer desired traits to essentially any plant. A wide variety of plants and plant cell lines can be engineered for the desired physiological and agricultural properties described herein using the nucleic acid constructs of the present disclosure and the various transformation methods described above. In certain embodiments, target plants and plant cells for manipulation are monocotyledonous and dicotyledonous plants such as cereals (eg wheat, corn, rice, millet, barley), fruit crops (eg tomatoes, apples, pears, Strawberries, oranges), forage crops (eg alfalfa), root crops (eg carrot, potato, sugar beet, yam), leafy crops (eg lettuce, spinach); flowering plants (eg petunia, rose, chrysanthemum) Conifers and pine trees (eg pine, fir, spruce); plants used in phytoremediation (environmental / soil purification by plants) (eg heavy metal accumulating plants); oil crops (eg sunflower, rapeseed) and for experimental purposes Including but not limited to plants used (eg, Arabidopsis)Accordingly, the disclosed methods and compositions include the genus Asparagus, Avena, Brassica, Citrus, Cytrulus and Watermelon. (Citrullus), Capsicum, Capsicum, Cucurbita, Daucus, Glycine, Gossypium, Hordeum (Hordeum), Lactuca, Lycopersicon, Malus, Malus, Manihot, Nicotiana, tobacco Nicotiana), Oryza, Persea, Persea, Pisum, Pils Pyrus, Prunus, Raphanus, Raphanus, Secale, Solanum, Sorghum, Sorghum ), Species from, but not limited to, Triticum, Triticum, Vitis, Vigna and Zea Has a wide range of uses.

  Those skilled in the art will recognize that once an expression cassette has been stably incorporated into a transgenic plant and confirmed to be operable, it can be introduced into other plants by sexual crossing. Depending on the species to be mated, any of a number of standard mating techniques can be used.

  Transformed plant cells, callus, tissue or plants can be identified and isolated by selecting or screening engineered plant material for traits encoded by marker genes present on the transforming DNA. For example, selection can be performed by growing engineered plant material in a medium containing an inhibitory amount of antibiotic or herbicide to which the transformed gene construct confers resistance. In addition, transformed plants and plant cells can also be identified by screening for the activity of any visible marker gene (eg, β-glucuronidase, luciferase, B or C1 gene) that may be present on the recombinant nucleic acid construct. . Such selection and screening methods are well known to those skilled in the art.

  Physical and biochemical methods can also be used to identify plants or plant cell transformants containing the inserted genetic construct. These methods include: 1) Southern analysis or PCR amplification to detect and determine the structure of the recombinant DNA insert; 2) Northern blotting to detect and examine RNA transcripts of gene constructs, S1 RN Ase protection, primer extension or reverse transcriptase PCR amplification; 3) if such a gene product is encoded by a gene construct, an enzyme assay to detect enzyme or ribozyme activity; 4) the product of the gene construct is a protein In some cases, including but not limited to protein gel electrophoresis, Western blot techniques, immunoprecipitation or solid phase enzyme immunoassay. Additional techniques such as in situ hybridization, enzyme staining and immunostaining can also be used to detect the presence or expression of recombinant constructs in specific plant organs and tissues. Methods for performing all these assays are well known to those skilled in the art.

  The effects of genetic manipulation using the methods disclosed herein can be observed, for example, by Northern blotting of RNA (eg, mRNA) isolated from the target tissue. Typically, if the amount of mRNA is increased, it can be assumed that the corresponding endogenous gene is expressed at a higher rate than before. Other methods of measuring gene and / or CYP74B activity can also be used. Depending on the substrate used and the method for detecting the increase or decrease in reaction products or by-products, various types of enzyme assays can be used. In addition, the level of expressed genes and / or CYP74B protein can be determined immunochemically, ie, ELISA, RIA, EIA and other antibody based assays well known to those skilled in the art, such as electrophoretic detection assays (staining or Western blots). Together with the method). The transgene may be selectively expressed in some tissues of the plant or at some developmental stages, or the transgene is expressed in substantially all plant tissues or substantially throughout its life cycle. obtain. However, any combinatorial expression pattern can be applied.

  The present disclosure also encompasses the seeds of the transgenic plants described above wherein the seed has a transgene or gene construct. The present disclosure further includes the progeny, clones, cell lines or cells of the transgenic plants described above having a transgene or gene construct.

  ZFPs and expression vectors encoding ZFPs can be administered directly to plants for targeted cleavage and / or recombination.

  Administration of an effective amount depends on any of the routes normally used to introduce ZFP and finally make contact with the plant cells to be treated. ZFP is administered by any suitable method. Suitable methods of administering such compositions are available and well known to those of skill in the art, but more than one route can be used to administer a particular composition, and a particular route is often more than another route. Can also provide a quicker and more effective reaction.

  A carrier can also be used, and the carrier can be determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions that can be used (see, eg, Remington's Pharmaceutical Sciences, 17th ed. 1985).

Applications Zinc finger proteins comprising one or more non-canonical zinc fingers as described herein include gene activation; gene repression; genome editing (cutting, target insertion, substitution or deletion); and epigenome editing (histone or Canonical C ₂ H ₂ ZFPs are useful for all currently used genomic regulatory and editing applications, including but not limited to (by targeting covalent modification of DNA).

  ZFNs comprising non-canonical zinc fingers as disclosed herein are DNA in a region of interest within cellular chromatin (eg, at a desired or predetermined site within a genome, eg, a mutated or wild type gene). Can be used for cutting. For such target DNA cleavage, a fusion protein comprising an engineered zinc finger binding domain and a cleavage domain, wherein the zinc finger binding domain is engineered to bind to the target site at or near a predetermined cleavage site Is expressed in cells. After the zinc finger portion of the fusion protein binds to the target site, the DNA is cleaved near the target site by the cleavage domain. The exact site of cleavage depends on the length of the ZC linker.

  Alternatively, two ZFNs, each containing a zinc finger binding domain and a cleavage half domain, are expressed in the cell, and the functional cleavage domain is reconstituted and binds to target sites juxtaposed such that the DNA is cleaved near the target site Let In one embodiment, cleavage occurs between the target sites of two zinc finger binding domains. One or both of the zinc finger binding domains can be manipulated.

  For targeted cleavage using a zinc finger binding domain-cleavage domain fusion polypeptide, the binding site can include a cleavage site or the proximal end of the binding site is 1, 2, 3, 4, 5, There may be 6, 10, 25, 50 or more nucleotides (or any integer value between 1 and 50 nucleotides). The exact location of the binding site relative to the cleavage site depends on the specific cleavage domain and the length of the ZC linker. For methods using two fusion polypeptides, each containing a zinc finger binding domain and a cleavage half domain, the binding site is generally located across the cleavage site. Thus, the proximal end of the first binding site is 1, 2, 3, 4, 5, 6, 10, 25 or more nucleotides (or some integer value between 1 and 50) on one side of the cleavage site. The proximal end of the second binding site is 1, 2, 3, 4, 5, 6, 10, 25 or more nucleotides (or 1-50 nucleotides) on the other side of the cleavage site. Any integer value between). Methods for mapping cleavage sites in vitro and in vivo are known to those skilled in the art.

  Once introduced into or expressed in the target cell, the fusion protein binds to the target sequence and cleaves at or near the target sequence. The exact site of cleavage depends on the nature of the cleavage domain and / or the presence and / or nature of the linker sequence between the binding domain and the cleavage domain. When using two ZFNs, each containing a truncated half domain, the distance between the proximal ends of the binding site is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25 or more Of nucleotides (or any integer value between 1 and 50). The optimal level of cleavage is also the distance between the binding sites of the two ZFNs (eg Smith et al. (2000) Nucleic Acids Res. 28: 3361-3369; Bibikova et al. (2001) Mol. Cell. Biol. 21 : 289-297) and the length of the ZC linker within each ZFN. See also US Patent Publication No. 20050064474A1 and International Publication Nos. WO05 / 084190, WO05 / 014791 and WO03 / 080809, the disclosures of which are incorporated herein by reference.

  Two ZFNs, each containing a truncated half domain, can bind within a region of interest of the same or opposite polarity, and their binding site (ie target site) can be any number of nucleotides, eg 0-50 nucleotides It can be separated by a pair or any integer value of nucleotide pairs in between. In certain embodiments, the binding sites for two fusion proteins each comprising a zinc finger binding domain and a cleavage half domain are 5-18 nucleotide pairs when measured from the end of each binding site closest to the other binding site. Can be located remotely, eg, 5-8 nucleotide pairs apart, or 15-18 nucleotide pairs apart, or 6 nucleotide pairs apart, or 16 nucleotide pairs apart, or within 10 nucleotide pairs of each other , Cleavage occurs between the binding sites.

  The site at which the DNA is cleaved is generally located between the binding sites for the two fusion proteins. DNA double-strand breaks often result from two single-strand breaks, or "nicks", offset by 1, 2, 3, 4, 5, 6 or more nucleotides (For example, cleavage of double-stranded DNA by native Fok I results from single-strand breaks offset by 4 nucleotides). Thus, cleavage does not necessarily occur at exactly the opposite site of each DNA strand. In addition, the distance between the structure of the fusion protein and the target site can affect whether cleavage occurs adjacent to one nucleotide pair or whether cleavage occurs at several sites. However, for many applications, including targeted recombination and targeted mutagenesis, cleavage within a stretch of nucleotides is generally sufficient and no cleavage between specific base pairs is necessary.

  As described above, the fusion protein can be expressed in a cell after introduction of the polypeptide and / or polynucleotide into the cell. For example, two polynucleotides each containing a sequence encoding one of the polypeptides can be introduced into a cell, and when the polypeptide is expressed and each binds to its target sequence, either at the target sequence or at that Disconnection occurs nearby. Alternatively, a single polynucleotide containing sequences encoding both fusion polypeptides is introduced into the cell. The polynucleotide can be DNA, RNA or any modified form or analog of DNA and / or RNA.

  In certain embodiments, targeted cleavage within a genomic region by ZFN results in a change in the nucleotide sequence of the region after repair of a cleavage event by non-homologous end joining (NHEJ).

  In other embodiments, target cleavage within a genomic region by ZFNs also results in a genomic sequence (eg, a region of interest within cellular chromatin) that is identical to a homology-dependent mechanism (eg, a predetermined genomic sequence (ie, a target site)). Or a homologous but non-identical sequence of one or more donor sequences that include exogenous sequences together with a non-identical sequence (ie by targeted recombination). . Double-strand breaks in cellular DNA stimulate cell repair mechanisms thousands of times in the vicinity of the cleavage site, so the targeted cleavage with ZFN described here results in sequence changes at virtually any site in the genome. Or allows substitution (by homology directed repair).

  Targeted replacement of selected genomic sequences requires the introduction of exogenous (donor) polynucleotides in addition to the ZFNs described herein. The donor polynucleotide can be introduced into the cell prior to, simultaneously with, or after expression of ZFN. The donor polynucleotide contains sufficient homology to the genomic sequence to support homologous recombination (or homology directed repair) between itself and the genomic sequence carrying homology. Sequence homology of about 25, 50, 100, 200, 500, 750, 1,000, 1,500, 2,000 nucleotides or more (or any integer value between 10 and 2,000 nucleotides or more ) Supports homologous recombination. Donor polynucleotides can range from 10 to 5,000 nucleotides (or some integer value in between) or longer.

  It is readily apparent that the nucleotide sequence of the donor polynucleotide is typically not identical to that of the genomic sequence it replaces. For example, the sequence of the donor polynucleotide can include one or more substitutions, insertions, deletions, inversions or rearrangements relative to the genomic sequence, so long as there is sufficient homology with the chromosomal sequence. Such sequence changes can be of any magnitude and can be as small as one nucleotide pair. Alternatively, the donor polynucleotide may comprise a non-homologous sequence (ie, an exogenous sequence to be distinguished from an exogenous polynucleotide) that is flanked by two regions of homology. In addition, the donor polynucleotide can include a vector molecule that includes a sequence that is not homologous to a region of interest within cellular chromatin. In general, the homologous region of the donor polynucleotide has at least 50% sequence identity with the genomic sequence desired for recombination. In certain embodiments, there is 60%, 70%, 80%, 90%, 95%, 98%, 99% or 99.9% sequence identity. Depending on the length of the donor polynucleotide, there can be any value of sequence identity between 1% and 100%.

  The donor molecule may contain several discontinuous regions that are homologous to cellular chromatin. For example, for targeted insertion of sequences that are not normally present in the region of interest, the sequences may be present in the donor nucleic acid molecule and flanked by regions that are homologous to sequences in the region of interest.

  To simplify assays that determine the success of sequence insertion from donor polynucleotides (eg, hybridization, PCR, restriction enzyme digestion), certain sequence differences may exist within the donor sequence compared to the genomic sequence. . Preferably, when located within the coding region, such nucleotide sequence differences do not alter the amino acid sequence or cause silent amino acid changes (ie, changes that do not affect the structure or function of the protein). The donor polynucleotide may optionally include changes in the sequence corresponding to the zinc finger domain binding site in the region of interest to prevent cleavage of the donor sequence introduced into the cellular chromatin by homologous recombination.

  A polynucleotide can be introduced into a cell as part of a vector molecule having additional sequences such as origins of replication, promoters and genes encoding antibiotic resistance. In addition, the donor polynucleotide can be introduced as a naked nucleic acid, as a nucleic acid coupled to a substance such as a liposome or poloxamer, or a bacterium or virus (eg, Agrobacterium, Rhizobium NGR234, Sinorizobium merillotti, Mesozobium rotii). Tobacco mosaic virus, potato X virus, cauliflower mosaic virus and cassava vein mosaic virus). See, for example, Chung et al. (2006) Trends Plant Sci. 11 (1): l-4.

  Due to changes in the chromosomal sequence, as long as enough donor sequence is copied to produce the desired sequence change, the full length sequence of the donor copied to the chromosome is not necessary.

  The efficiency of insertion of the donor sequence by homologous recombination is inversely related to the distance between the double-strand break and the site where recombination is desired in cellular DNA. In other words, higher homologous recombination efficiency is observed when the double-strand break is closer to the site where recombination is desired. If the exact site of recombination is not predetermined (eg, the desired recombination event can occur over the distance of the genomic sequence), the length and sequence of the donor nucleic acid, along with the cleavage site, will yield the desired recombination event. Selected. When the desired event is designed to change the sequence of one nucleotide pair within the genomic sequence, cellular chromatin is cleaved within 10,000 nucleotides on either side of the nucleotide pair. In certain embodiments, 1,000, 500, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, on either side of the nucleotide pair whose sequence is to be altered. Cleavage occurs within 5 or 2 nucleotides, or some integer value between 2 and 1,000.

  Targeted insertion of exogenous sequences into the genomic region is accomplished by targeted cleavage within the genomic region using ZFNs in conjunction with providing an exogenous (donor) polynucleotide containing the exogenous sequence. The donor polynucleotide also typically includes a sequence adjacent to the exogenous sequence, including sufficient homology to the genomic region to support double-strand break homology-directed repair within the genomic sequence, and To insert exogenous sequences into the genomic region. Thus, the donor nucleic acid can be any size sufficient to support the integration of exogenous sequences by homology-dependent repair mechanisms (eg, homologous recombination). While not wishing to be bound by any particular theory, a region of homology adjacent to an exogenous sequence provides a template for resynthesis of the genetic information of the double-strand break site at the end of the broken chromosome. Seem.

  Targeted integration of exogenous sequences, as described above, can be used to insert marker genes at selected chromosomal locations. Marker genes are sequences encoding proteins that mediate antibiotic resistance (eg ampicillin resistance, neomycin resistance, G418 resistance, puromycin resistance), colored or fluorescent or luminescent proteins (eg green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein) Protein, luciferase) and sequences encoding proteins that mediate cell growth enhancement and / or gene amplification enhancement (eg, dihydrofolate reductase). Exemplary marker genes are therefore β-glucuronidase (GUS), phosphinothricin N-acetyltransferase (PAT, BAR), neomycin phosphotransferase, β-lactamase, catechol dioxygenase, α-amylase, tyrosinase, β-galactosidase , Luciferase, aequorin, EPSP synthase, nitrilase, acetolactate synthase (ALS), dihydrofolate reductase (DHFR), dalapon dehalogenase and anthranilate synthase. In certain embodiments, targeted integration is used to insert sequences responsible for the regulated expression of an RNA expression construct, eg, microRNA or siRNA. Promoters, enhancers and additional transcriptional regulatory sequences can also be incorporated into the RNA expression construct.

In cells comprising a zinc finger / nuclease fusion molecule and a donor DNA molecule, a further increase in the efficiency of targeted recombination, homologous drive repair process is active maximally blocking the cells in the G ₂ phase of the cell cycle Achieved by: Such a stop can be realized in many ways. For example the cells, for example, drugs that affect cell cycle progression such that cells stop at G ₂ phase can be treated with the compounds and / or low molecular. Exemplary molecules of this type include compounds that affect microtubule polymerization (eg, vinblastine, nocodazole, taxol), compounds that interact with DNA (eg, cisplatinum (II) dichloride diamine, cisplatin, doxorubicin) and / or Examples include, but are not limited to, compounds that affect DNA synthesis (eg, thymidine, hydroxyurea, L-mimosine, etoposide, 5-fluorouracil). Further increase in recombination efficiency is due to the use of histone deacetylase (HDAC) inhibitors (eg, sodium butyrate, trichostatin A) that alter chromatin structure to make genomic DNA more accessible to the cellular recombination machinery Achieved by:

  Further methods for cell cycle arrest include overexpression of proteins that inhibit the activity of CDK cell cycle kinases, eg by introducing cDNA encoding the protein into the cell or activating expression of the gene encoding the protein Over-expression of said protein by introducing into the cell an engineered ZFP. Cell cycle arrest can also be achieved by inhibiting the activity of cyclins and CDKs using, for example, RNAi methods (eg, US Pat. No. 6,506,559) or one or more genes involved in cell cycle progression, For example, it can also be achieved by introducing into a cell an engineered ZFP that suppresses the expression of cyclin and / or CDK gene. See, eg, co-owned US Pat. No. 6,534,261 for methods for the synthesis of engineered zinc finger proteins for regulation of gene expression.

  As noted above, the disclosed methods and compositions for targeted cleavage can be used to induce mutations within genomic sequences. Target cleavage can also be used to create gene knockouts (eg, functional genomics or target validation) and to facilitate targeted insertion of sequences into the genome (ie, gene knockin). Insertion is performed by replacing a chromosomal sequence through homologous recombination, or by inserting a new sequence (ie, a sequence not existing in the target region) flanked by a sequence homologous to the target region in the chromosome at a predetermined target site. By targeted integration. The same method can also be used to replace a wild type sequence with a mutant sequence or to convert one allele to a different allele.

  Targeted cleavage of an infected or integrated plant pathogen can be used to treat a pathogen infection in a plant host, for example by cleaving the genome of the pathogen such that its pathogenicity is reduced or eliminated. In addition, targeted cleavage of genes encoding receptors for plant viruses can be used to block expression of such receptors, thereby preventing viral infection and / or virus spread in plants.

  Exemplary plant pathogens include plant viruses such as Alfamoviruses, Alphacryptoviruses, Badnaviruses, Betacryptoviruses, Bigeminiviruses, Bromoviruses, Bymoviruses, Capilloviruses, Carlaviruses, Carmoviruses, Caulimoviruses, Closeroviruses, Comoviruses, Cucumoviruses ), Cytohabdoviruses, Dianthoviruses, Enamoviruses, Fabaviruses, Fijiviruses, Furoviruses, Holday Virus (Hordeiviruses), Hybridigeminiviruses, Idaeoviruses, Ilarviruses, Ipomoviruses, Luteoviruses, Macromoviruses, Macluraviruses, Malafiviruses, Monogeminiviruses, Nanaviruses, Necroviruses, Nepoviruses, Nucleorhabdoviruses, Orizaviruses, Urmiaviruses, Urmiaviruses Phytoreoviruses, Potexviruses, Potyviruses, Rymoviruses, Satellite RNA, Satellite viruses, Seki Irus (Sequiviruses), Sobemovirus (Sobemoviruses), Tenuiviruses (Tenuiviruses), Tobamoviruses, Tobraviruses (Tobraviruses), Tombusviruses, Tospoviruses, Toripoviruses, Trichoviruses, Timovirus ( Tymoviruses), Umbraviruses, Baricosaviruses and Waikaviruses; fungal pathogens such as smut fungi (eg Ustilaginales), rust fungi (Uredinales), ergots ( Clavicepts pupurea and powdery mildews; filamentous fungi (Oomycetes) such as Phytophthora infestans (potato blight); bacterial pathogens such as Erwinia E. herbicola), Pseudomonas (eg, P. aeruginosa), P. syringae, P. fluorescense and P. putida (P. putida), Ralstonia (eg R. solanacearum, Agrobacterium and Xanthomonas; roundworms (Nematoda); and Phytomyxea (Polymyxa) And Plasmodiophora), but is not limited thereto.

  The disclosed methods for targeted recombination can be used to replace any genomic sequence with a homologous non-identical sequence. For example, a mutated genomic sequence can be replaced by its wild-type counterpart, thereby providing a method for the treatment of plant diseases; conferring resistance to plant pathogens; increasing crop yields, etc. Is possible. Similarly, one allele of a gene can be replaced by a different allele using the methods of targeted recombination disclosed herein.

  In many of these cases, the region of interest contains a mutation and the donor polynucleotide contains the corresponding wild type sequence. Similarly, the wild type genomic sequence can be replaced by a mutated sequence if it is desired. Indeed, diseases that depend in some way on specific genomic sequences can be repaired or alleviated using the methods and compositions disclosed herein.

  Target cleavage and targeted recombination can also be used to alter non-coding sequences (eg regulatory sequences such as promoters, enhancers, initiators, terminators, splice sites) to alter the level of expression of the gene product. Such methods can be used, for example, for therapeutic purposes, changes in cell physiology and biochemistry, functional genomics and / or target validation tests.

  The methods and compositions described herein can also be used for activation and suppression of gene expression using fusions between non-canonical zinc finger binding domains and functional domains. Such methods are disclosed, for example, in co-owned US Pat. Nos. 6,534,261; 6,824,978 and 6,933,113, the disclosures of which are incorporated herein by reference. ing. Additional methods of suppression include the use of antisense oligonucleotides and / or small interfering RNA (siRNA or RNAi) targeted against the sequence of the gene to be suppressed.

  In an additional embodiment, in addition to or instead of the zinc finger cleavage domain fusion disclosed herein, to facilitate targeted recombination, between the zinc finger binding domain and the recombinase (or functional fragment thereof). One or more fusions can be used. See, for example, commonly owned US Pat. No. 6,534,261 and Akopian et al. (2003) Proc. Natl. Acad. Sci. USA 100: 8688-8691.

  In additional embodiments, the disclosed methods and compositions comprise transcriptional activation or repression domains and ZFP binding that require dimerization (homodimerization or heterodimerization) for activity. Used to provide a fusion of domains. In these cases, the fusion polypeptide comprises a zinc finger binding domain and a functional domain monomer (eg, a monomer from a dimer transcriptional activation or repression domain) virus. Binding of such two fusion polypeptides to a properly located target site allows dimerization to reconstitute functional transcriptional activation or repression domains.

  The invention is further defined in the following examples, in which all percentages and percentages are by weight and temperatures are in degrees Celsius unless otherwise specified. While these examples illustrate certain embodiments of the present invention, it should be understood that they are provided as an illustration only. From the above considerations and these examples, those skilled in the art can ascertain the basic features of the present invention and to adapt the present invention to various applications and conditions without departing from the spirit and scope thereof. Various changes and modifications of the invention can be made.

ZFN expression vectors The four-finger ZFN ("" Expression vectors containing sequences encoding 5-8 "and" 5-9 ") were modified as follows. Briefly, 5-8 and 5-9ZFN (a nuclease domain of type IIS restriction enzyme FokI by a 4 amino acid ZC linker (Wah et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10564-10569). The four zinc finger domains fused to amino acids 384-579) of the sequence) were modified to a CCHC structure. Further modifications (substitutions and insertions) were also made on the residues between the C-terminal His and Cys zinc coordination structures and / or finger 2 and / or finger 4 on the C-terminal side of the C-terminal Cys.

Gene repair of eGFP in reporter cell lines The ability of ZFNs containing the CCHC zinc fingers described herein to promote homologous recombination has been demonstrated by Urnov (2005) Nature 435 (7042): 646-51 and US Patent Publication No. 20050064474 (eg, Tested in the GFP system described in Examples 6-11). Briefly, 50 ng of each ZFN and 500 ng of promoterless GFP donor (Urnov (2005) Nature) were transferred to 500,000 reporter cells using 2 μL of Lipofectamine 2000 per sample according to the Invitrogen Lipofectamine 2000 protocol. I did it.

  Vinblastine was added at a final concentration of 0.2 μM 24 hours after transfection and removed 72 hours after transfection.

  Cells were assayed for GFP expression 5 days after transfection by measuring 40,000 cells for each transfection on a Guava tabletop FACS analyzer.

  As shown in FIG. 1, most ZFNs containing the altered CCHC zinc fingers shown in Tables 1 and 2 above promote homologous recombination at the reporter (GFP) locus and are levels above the unmodified CCHC zinc fingers. Of GFP expression, some of which were comparable in performance to ZFNs containing CCHH zinc fingers. A variant with optimal performance when positioned on finger 4 (F4) includes the following sequence (including His zinc coordination residue and its C-terminal side): HAQRCGLRGSQLV (SEQ ID NO: 53) (referred to as No. 21 in Table 2) 1 and zinc fingers indicated as “2-21” in FIG. A variant with optimal performance when positioned on finger 2 (F2) includes the following sequence (including His zinc coordination residue and its C-terminal side): HIRTCTGSQKP (SEQ ID NO: 75) (referred to as No. 43 in Table 2) 1 and zinc fingers indicated as “2-43” in FIG.

Editing of the chromosomal IL2Rγ gene by targeted recombination The ZFN described here is an endogenous IL2Rγ assay described in Urnov (2005) Nature 435 (7042): 646-51 and Example 2 of US Patent Publication No. 20050064474. Also tested. Briefly, 2.5 μg of each ZFN expression construct was transfected into 500,000 K562 cells using a Nucleofector (Amaxa). Genomic DNA was collected and assayed for gene disruption at the endogenous IL2Rγ locus using the Surveyor endonuclease kit.

  ZFN is shown in the upper left of FIG. In particular, altered zinc finger 20 refers to a CCHC zinc finger comprising the sequence HTRRCGLRGSQLV; zinc finger 21 comprises the sequence HAQRCGLRGSQLV (SEQ ID NO: 53); zinc finger 43 comprises the sequence HIRTCTGSQKP (SEQ ID NO: 75); zinc finger 45 Contains the sequence HIRTCGCTGSQKP; zinc finger 47 contains the sequence HIRCTGGSQKP; and zinc finger 48 contains the sequence HIRRCGCTGSQKP. Zinc fingers 20 and 21 were used on finger 4 of 4-finger ZFN, and zinc fingers 43, 45, 47 and 48 were used on finger 2 of 4-finger ZFN.

The tested ZFN pairs are shown in FIG.

  To determine if the mutation was induced at the cleavage site, the amplification product was analyzed using the Cel-1 assay, which was denatured and renatured and then treated with a mismatch specific Cel-1 nuclease. See, for example, Oleykowski et al (1998) Nucleic Acids res. 26: 4597-4602; Qui et al. (2004) BioTechniques 36: 702-707; Yeung et al. (2005) BioTechniques 38: 749-758.

  The results of two experiments are shown for each sample in FIG. Experiment No. 8 for Samples 8 and 9 2 had significant background noise in the lane, which reduced the apparent effect of these ZFNs.

As shown in FIG. 2, some CCHC variants were essentially equivalent to wild type C ₂ H ₂ ZFN. Finger 4 zinc finger 21 (samples 5 and 9) produced better results than finger 4 zinc finger 20 (samples 4 and 8). In finger 2, zinc finger 43 produced the best results.

Gene repair of eGFP in a reporter cell line Based on the results shown in FIGS. 1 and 2, CCHC zinc fingers (referred to as 1a to 10a) shown in Tables 3 and 4 were prepared. These zinc fingers were incorporated into 5-8 and 5-9 ZFNs and tested in the GFP gene repair assay described in Example 2 above. The ZFN pairs tested in each sample are shown below each bar, with zinc finger numbers 20, 21, 43, 45, 47 and 48 as described in Example 3 and CCHC zinc fingers 1a-10a. Includes the sequences shown in Tables 3 and 4 above. Zinc fingers 20, 21, 7a, 8a, 9a and 10a were used on finger 4; zinc fingers 43, 45, 47, 48, 1a, 2a, 3a, 4a, 5a and 6a were used on finger 2.

  The results are shown in FIG. The top row at the bottom of each bar represents the zinc fingers incorporated into ZFN 5-8, and the bottom row of each bar represents the zinc fingers incorporated into ZFN 5-9. For example, the second bar from the left of the graph of FIG. 3 represents samples transfected with 5-8 and 5-9 ZFN, where F4 of both ZFNs contains the zinc finger 20 sequence. As shown, many of the ZFNs containing CCHC zinc fingers performed as well as wild type (CCHH) ZFNs.

Target vector design and production Overall structure of the target sequence The target construct for tobacco (dicotyledonous plants) contained the following seven components shown in FIGS. i) A. Tumefaciens open reading frame 24 (orf-24) 3 ′ untranslated region (UTR) (Gelvin et al., 1987, European Patent No. EP222493), E. coli HPT gene (Waldron et al., 1985, Hygrins containing Arabidopsis ubiquitin-3 (ubi-3) promoter (Callis, et al., 1990, J. Biol. Chem. 265-12486-12493) driving Plant Mol. Biol. 18: 189-200) Mycin phosphotransferase (HPT) expression cassette; ii) N. H. homologous sequence-1 including N. Tabacum RB7 matrix binding region (MAR) (Thompson et al., 1997, International Publication No. WO 9727207); iii) Modified A. 5 'Green Fluorescent Protein (GFP) Gene Fragment Driven by the Tumefaciens Mannopine Synthase (Δmas) Promoter (Petolino et al., US Pat. No. 6,730,824) (Evrogen Joint Stock Company, Moscow, Russia) Iv) A. Β-glucuronidase (GUS) gene (Jefferson, 1987, terminated by the tumefaciens nopaline synthase (nos) 3′UTR (DePicker et al., 1982, J. Mol. Appl. Genet. 1: 561-573) GUS expression cassette containing cassava vein mosaic virus (CsVMV) promoter (Verdaguer et al., 1996, Plant Molecular Biology 31: 1129-1139) driving Plant Mol. Biol. Rep. 5: 387-405); v) A. 3'GFP gene fragment (Evrogen Joint Stock Company, Moscow, Russia) terminated by tumefaciens orf-1 3'UTR (Huang et al., J. Bacteriol. 172: 1814-1822); vi) Arabidopsis 4-coumaroyl -Homologous sequence-2 including CoA synthase (4-CoAS) intron-1 (locus At3g21320, GenBank NC 003074) and vii) A. S. tumefaciens ORF-25 / 26 3′UTR (Gelvin et al., 1987, European Patent No. EP222493) S. viridochromogenes phosphinothricin phosphotransferase (PAT) (Wohlleben et al., 1988, Gene 70: 25-37) 3 ′ gene fragment.

  The zinc finger-Fok1 fusion protein binding site (IL-1-L0-Fok1) (Urnov et al., 2005, US 2005/0064474) was replaced with the CsVMV promoter (Verdaguer et al., 1996, Plant Molecular Biology 31: 1129 -1139) and fused with the GUS coding sequence (Jefferson, 1987, Plant Mol. Biol. Rep. 5: 387-405) at the N-terminus. Two copies of the second zinc finger-Fok1 fusion protein binding site (Scd27-L0-Fok1) (Urnov et al., 2005, US 2005/0064474) flanked the 5 'and 3' GFP gene fragments (Evrogen Joint Stock Company, Moscow, Russia). Since each binding site contained 4 tandem repeats of the recognition sequence of a specific zinc finger-Fok1 fusion protein, each binding site was ˜200 bp in size (FIG. 6A). This was designed to ensure that the recognition sequence is accessible to the zinc finger-Fok1 fusion protein in a complex chromatin environment. Each recognition sequence contained an inverted repeat sequence in which one zinc finger-Fok1 fusion protein was bound as a homodimer and cleaved double stranded DNA (FIG. 6B). In order to ensure that no functional GFP translation from the target sequence occurs, 5 'and 3' GFP gene fragments that overlap by 540 bp and homologous within the target sequence and a termination codon 3 of the 5 'GFP fragment. 'Insert at the end.

  A transformation vector containing the target sequence was generated through the multi-step cloning process described below.

B. Construction of HPT binary vector (pDAB1584) Arabidopsis driving the GUS gene (Jefferson, 1987, Plant Mol. Biol. Rep. 5: 387-405), terminated by the tumefaciens orf-1 UTR (Huang et al., J. Bacteriol. 172: 1814-1822) The vector pDAB1400 containing the GUS expression cassette containing the ubi-3 promoter (Callis, et al., 1990, J. Biol. Chem. 265-12486-12493) was used as the starting base construct (FIG. 7).

  In order to avoid unnecessary repetitive regulatory elements in the target construct, A. Tumefaciens orf-1 UTR (Huang et al., J. Bacteriol. 172: 1814-1822) was excised from pDAB782 (Figure 8) as a SacI / XbaI fragment and cloned into the same site in pDAB1400. Substitution with Tumefaciens orf-24 UTR (Gelvin et al., 1987, European Patent No. EP222493). The resulting construct is A. Arabidopsis ubi-, which drives the GUS gene (Jefferson, 1987, Plant Mol. Biol. Rep. 5: 387-405), terminated by the Tumefaciens orf-24 UTR (Gelvin et al., 1987, European Patent No. EP222493) It contains 3 promoters (Callis, et al., 1990, J. Biol. Chem. 265-12486-12493) and was named pDAB1582 (FIG. 9).

  The HPT coding sequence (Waldron et al., 1985, Plant Mol. Biol. 18: 189-200) was PCR amplified from pDAB354 plasmid (FIG. 10) using primers P1 and P2. A BbsI site was added to the 5 'end of primer P1 and a SacI site was retained at the 3' end of primer P2. The HPTII PCR fragment was digested with BbsI / SacI and cloned into pDAB1582 digested with NcoI-SacI to replace the GUS gene with the HPT gene from the PCR fragment. The resulting plasmid was named pDAB1583 (FIG. 11).

  Arabidopsis ubi-3 / HPT / A. The tumefaciens orf-24 fragment was then excised from pDAB1583 by NotI digestion and treated with T4 DNA polymerase to generate blunt ends. The blunt-ended HPT expression cassette was cloned into the binary base vector, pDAB2407 (FIG. 12) at the PmeI site, generating plasmid pDAB1584 (FIG. 13).

C. Construction of Vector (pDAB1580) Containing Homologous Sequence and Scd27 Zinc Finger-Fok1 Fusion Protein Binding Site To avoid repetitive regulatory sequences in the target vector, A. Tumefaciens orf-1 UTR (Huang et al., J. Bacteriol. 172: 1814-1822) Tumefaciens orf25 / 26UTR (Gelvin et al., 1987, European Patent No. EP222493). To perform UTR exchange, A. Tumefaciens orf25 / 26UTR (Gelvin et al., 1987, European Patent No. EP222493) was PCR amplified from pDAB4045 plasmid (FIG. 15) using primers P3 and P4. SmaI and AgeI sites were added to the 3 ′ end of the PCR fragment and the SacI site was retained at the 5 ′ end. A. Arabidopsis thaliana ubiquitin-10 driving the PAT gene (Wohlleben et al., 1988, Gene 70: 25-37), terminated by the tumefaciens orf-1 UTR (Huang et al., J. Bacteriol. 172: 1814-1822) (Ubi-10) promoter (Callis, et al., 1990, J. Biol. Chem. 265-12486-12493) and N.I. PDAB2418 plasmid DNA containing the PAT gene expression cassette containing the N. tabacum RB7 MAR sequence (Thompson et al., 1997, International Publication No. WO9727207) was digested with SacI and AgeI and the two largest fragments Was recovered. These fragments were digested with SacI and AgeI. It was ligated with the PCR product of Tumefaciens orf25 / 26UTR (Gelvin et al., 1987, European Patent No. EP222493). The resulting plasmid was named pDAB1575 (FIG. 16). N. Tabacam RB7 MAR (Thompson et al., 1997, WO 9727207) is used as homologous sequence-1 in the target vector.

  Intron-1 of Arabidopsis 4-CoAS (locus At3g21320, GenBank NC 003074) was selected for use as homologous sequence-2 in the target vector. The PAT gene (Wohlleben et al., 1988, Gene 70: 25-37) coding sequence was analyzed and introns were inserted 299/300 bp downstream of the start codon so that appropriate 5 ′ and 3 ′ splicing sites were formed. It was identified as a site to do. The full-length intron was then fused by DNA synthesis (Picoscript Ltd., LLP, Houston, Texas) with 253 bp of the 3 'partial PAT coding sequence. NotI and SacI sites were added to the 5 'and 3' ends of the DNA fragment, respectively. The synthesized DNA fragment was then digested with NotI / SacI and inserted into pDAB1575 at the same site to replace the full length PAT coding sequence. The resulting construct was named pDAB1577 (FIG. 17).

  A 241 bp DNA fragment containing four tandem repeats of the Scd27-L0-Fok1 recognition site (FIG. 6) was synthesized with SmaI sites added at both the 5 ′ and 3 ′ ends of the fragment (Picoscript Ltd., LLP, Houston, Texas). The synthesized zinc finger-Fok1 binding site-containing fragment was then digested with SmaI and inserted into pDAB1577 at the MscI site. The resulting vector was named pDAB1579 (FIG. 18). A second SmaI digested zinc finger-Fok1 binding site containing fragment was then inserted into pDAB1579 at the SwaI site. The resulting construct was named pDAB1580 (FIG. 19). This vector contains two synthetic Scd27 zincs containing homologous sequences 1 and 2 (N. tabacam RB7 MAR and Arabidopsis 4-CoAS intron-1 respectively) and four tandem repeats of each of the Scd27-L0-Fok1 recognition sites. Contains a finger-Fok1 binding site.

D. Construction of a vector containing two partially overlapping non-functional GFP fragments (pDAB1572) The GFP gene, CopGFP, was purchased from Evogen Joint Stock Company (Moscow, Russia) and the full-length coding sequence was used with primers P5 and P6 PCR amplification was performed. BbsI and SacI sites were added to the 5 ′ and 3 ′ ends of the PCR product, respectively. The CopGFP PCR product is then digested with BbsI / SacI at the NcoI / SacI site to replace the GUS gene. Drives GUS gene (Jefferson, 1987, Plant Mol. Biol. Rep. 5: 387-405), terminated by Tumefaciens orf-1 3′UTR (Huang et al., J. Bacteriol. 172: 1814-1822) Modification A. It was cloned into pDAB3401 (FIG. 20) containing the tumefaciens Δmas promoter (Petolino et al., US Pat. No. 6,730,824). The resulting vector was named pDAB1570 (FIG. 21).

  In order to generate two partially overlapping non-functional GFP fragments, a DNA fragment containing a majority of the CopGFP coding sequence and a 47 bp deletion at the 5 'end was PCR amplified using primers P9 and P10. An ApaI site was added to both the 5 'and 3' ends and an additional StuI site was added to the 5 'end downstream of the ApaI site. The PCR product was then digested with ApaI and inserted into pDAB1570 at the ApaI site, thereby creating two non-functional GFP fragments within the same vector with a 540 bp overlapping sequence. The resulting construct was named pDAB1572 (FIG. 22).

E. Construction of a Vector (pDAB1573) Containing IL-1 Zinc Finger-Fok1 Fusion Protein Binding Site / GUS Gene Fusion A 233 bp DNA fragment containing four tandem repeats of the IL-1_L0-Fok1 recognition site (FIG. 6) is 5 NcoI and AflIII sites were added to the 'terminal and 3' ends, respectively. , LLP, (Houston, Texas). The synthesized fragment is then digested with NcoI / AflIII, and A.I. Tumefaciens orf-1 3'UTR (Huang et al., J. Bacteriol. 172: 1814-1822) terminated and driven by the CsVMV promoter (Verdaguer et al., 1996, Plant Molecular Biology 31: 1129-1139) It was inserted into pDAB4003 (FIG. 23) containing the GUS gene (Jefferson, 1987, Plant Mol. Biol. Rep. 5: 387-405). Subsequently, an N-terminal fusion between the IL-1_Lo-Fok1 binding site and the GUS coding sequence was made. The resulting vector was named pDAB1571 (FIG. 24).

  To avoid repetitive 3'UTR elements in the target vector, C. tumefaciens nos 3'UTR (DePicker et al., 1982, J. Mol. Appl. Genet. 1: 561-573) was excised from pDAB7204 (Figure 25) as a SacI / PmeI fragment. To replace the tumefaciens orf-1 3'UTR (Huang et al., J. Bacteriol. 172: 1814-1822), it was cloned into pDAB1571 digested with SacI / NaeI. The resulting plasmid was named pDAB1573 (FIG. 26).

F. Construction of Final Target Vector (pDAB1585) To create the final target vector, the GUS expression cassette with IL-1-Fok1 fusion protein target site insertion was excised from pDAB1573 by NotI digestion, blunt ended, and pDAB1572 at the StuI site. Inserted. The resulting intermediate vector was named pDAB1574 (FIG. 27). Modified Δmas promoter (Petolino et al., US Pat. No. 6,730,824), 5 ′ partially overlapping GFP sequence (Evrogen Joint Stock Company, Moscow, Russia), CsVMV promoter (Verdaguer et al., 1996, Plant Molecular Biology 31: 1129- 1139), IL-1-Fok1 fusion protein target sequence, GUS gene (Jefferson, 1987, Plant Mol. Biol. Rep. 5: 387-405) coding region; Tumefaciens nos 3′UTR (DePicker et al., 1982, J. Mol. Appl. Genet. 1: 561-573), 3 ′ partially overlapping GFP (Evrogen Joint Stock Company, Moscow, Russia) and The entire cassette containing the tumefaciens orf-1 3′UTR (Huang et al., J. Bacteriol. 172: 1814-1822) was excised from pDAB1574 and inserted into pDAB1580 at the NotI site. The resulting plasmid was named pDAB1581 (FIG. 28). The AgeI fragment of pDAB1581 was then inserted into pDAB1584 at the AgeI site, thereby creating the final target construct pDAB1585 (FIGS. 4 and 5).

Generation of transgenic cell line with integrated target sequence A tobacco cell suspension culture, designated BY2, was used in which the target sequence of Example 5 was stably integrated by Agrobacterium transformation. The basal cell line, BY2, was obtained from Jun Ueki of Japan Tobacco, Iwata, Shizuoka, Japan. This culture grows as 5-10 μ diameter cells in 100-150 cell clusters with a doubling time of about 18 hours. BY2 cell suspension cultures were prepared from LS basic salt (PhytoTechnology Labs L689), 170 mg / L KH ₂ PO ₄ , 30 g / L sucrose, 0.2 mg / L 2,4-D and 0.6 mg / L thiamine-HCL, Maintained in medium containing pH 6.0. BY2 cells were subcultured every 7 days by adding 50 mL of LS basal medium to 0.25 mL of PCV. The BY2 cell suspension culture was maintained in a 250 mL flask on a rotary shaker at 25 ° C. and 125 RPM.

To generate a transgenic BY2 cell culture with integrated target sequence, the flask of tobacco suspension 4 days after subculture was divided into 10-12 4 mL aliquots, which were divided into 100 × 25 mm Petri dishes. in _were co-cultured with Agrobacterium strain LBA4404 100μL for holding pDAB1585 grown to OD 600 to 1.5 overnight. The dish was wrapped in parafilm and incubated for 3 days at 25 ° C. without shaking, after which the excess liquid was removed and replaced with 11 mL LS basal medium containing 500 mg / L carbenicillin.

  After resuspension of tobacco cells, 1 mL of the suspension is dispensed into 100 × 25 mm plates of appropriate basal medium containing 500 mg / L carbenicillin and 200 mg / L hygromycin coagulated with 8 g / L TC agar, Incubate at 28 ° C in the dark without wrapping with wrap. This resulted in 120-144 selection plates per treatment. Individual hygromycin resistant isolates appear 10-14 days after inoculation and are transferred to individual 60 × 20 mm plates (one isolate per plate) for analysis and subsequent retransformation experiments. The callus was maintained on a 14-day subculture schedule until needed.

Screening and characterization of target transgenic events The hygromycin resistant transgenic events resulting from transformation of the target vector into BY2 tobacco cell culture described in Example 6 were analyzed as follows.

  Initial analyzes performed to screen for these transgenic events include GUS expression analysis to show target sequence accessibility, partial and full-length target sequence PCR to confirm the presence and integrity of the target vector Analysis and Southern blot analysis to determine the copy number of the incorporated target sequence was included. A subset of transgenic events that showed GUS expression included a single copy of the full-length target sequence; these were used to re-establish suspension cultures to generate target lines for subsequent retransformation. Selected. These re-established target lines were subjected to further characterization, which included more detailed Southern blot analysis, sequencing confirmation of the entire target insert and flanking genomic sequence analysis.

  Suspension cultures initiated from transgenic tobacco callus tissue or selection events were analyzed for GUS activity by incubating 50 mg of sample in 150 μL of assay buffer at 37 ° C. for 24-48 hours. Assay buffer was 0.2 M sodium phosphate pH 8.0, 0.1 mM potassium ferricyanide and potassium ferrocyanide, 1.0 mM sodium EDTA, 0.5 mg / mL 5-bromo-4-chloro-3-indoyl-β, respectively. -Glucuronide and 0.6% (v / v) Triton X-100 (Jefferson, 1987, Plant Mol. Biol. Rep. 5: 387-405). The appearance of the blue colored region was used as an indicator of GUS gene expression, which indicated that the target sequence insertion was transcriptionally active and therefore accessible in the local genomic environment.

  A primer pair that directs a transgenic event expressing GUS to amplify a 10 kb DNA fragment spanning from the 3′UTR of the HPT expression cassette at the 5 ′ end of the target sequence to the 3′UTR of the partial PAT gene cassette at the 3 ′ end of the target sequence Assayed by PCR using P15 / P16. Since all events were obtained under hygromycin selection, it was assumed that the HPT expression cassette was intact in all target events. Therefore, only the 3'UTR of the HPT expression cassette was considered for full length PCR analysis. A subset of events was also PCR assayed using primer pairs P15 / P17 and P18 / P19, respectively, to determine the integrity of the 5 'and 3' ends of the target sequence. In order to determine the copy number of the incorporated target sequence, all target events confirmed by PCR analysis were further assayed by Southern blot analysis.

  Southern blot analysis was performed on all target events that passed GUS expression and full-length PCR screening. Ten μg of genomic DNA was digested with NsiI, the only cutter enzyme in the target sequence. Digested genomic DNA was separated on a 0.8% agarose gel and transferred to a nylon membrane. After crosslinking, the DNA transferred to the membrane was hybridized with an HPT gene probe in order to determine the copy number at the 5 'end of the target sequence. The same blot was then stripped and rehybridized with the PAT gene probe to determine the copy number at the 3 'end of the target sequence.

A number of events that showed GUS expression and contained one copy of the full-length target sequence were selected for further characterization, including more detailed Southern blot analysis, confirmation of the entire target sequence and adjacent genomic sequence analysis. One event called BY2-380 was selected based on molecular characterization. Suspension cultures were re-established from the event for subsequent re-transformation with vectors containing donor DNA and non-C ₂ H ₂ zinc finger -Fok1 fusion protein gene.

  To ensure that the suspension culture established from target event BY2-380 contains the intact target sequence as expected, the target sequence 3'UTR from the 3'UTR of the HPT expression cassette at the 5'end of the target sequence The major target sequence up to the 3'UTR of the 'terminal partial PAT gene cassette was PCR amplified using primer pair P15 / P16 and cloned into the pCR2.1 TOPO vector (Invitrogen, Carlsbad, California). The PCR product inserted into the TOPO vector is described in Lark technology, Inc. (Hoouston, Texas). The sequencing results indicated that BY2-380 has the complete target sequence as expected.

  The BY2-380 cell line was further analyzed to obtain flanking genomic sequences using the Universal GenomeWalker Kit (Clontech, Mountain View, California). Briefly, 2.5 μg genomic DNA was digested with three blunt end restriction enzymes, EcoRV, DraI and StuI in separate reactions. The digested DNA was purified through phenol / chloroform extraction and ligated with a BD GenomeWalker Adapter. Nested PCR amplification, using the ligation as a template, primers P20 (upstream walking at the 5 ′ end of target sequence insertion) and P21 (downstream walking at the 3 ′ end of target sequence insertion) and secondary nested for the primary PCR reaction For the PCR reaction, primers P22 (upstream walking at the 5 ′ end of target sequence insertion) and P23 (downstream walking at the 3 ′ end of target sequence insertion) were used. The amplified fragment from the secondary PCR reaction was cloned into pCR2.1 TOPO or pCR Blunt II TOPO vector (Invitrogen, Carlsbad, California) and determined using Dye Terminator Cycle Sequencing Kit (Beckman Coulter, Fuller). Through this process, flanking genomic sequences were obtained from the BY2-380 target line. Primers were then digested based on adjacent genomic sequences and used to amplify the entire target sequence.

  The amplified fragment obtained from this target line was the expected size. Both ends of the amplified fragment were confirmed by sequencing.

Design and construction of donor DNA vectors Donor DNA constructs include homologous sequence-1 (N. tabacum RB7 MAR) (Thompson et al., 1997, International Publication No. WO97727207), full-length Arabidopsis ubi-10 promoter (Callis, et al., 1990, J. Biol. Chem. 265-12486-12493), 299 bp of 5 'partial PAT gene coding sequence (Wohlleben et al., 1988, Gene 70: 25-37) and homologous sequence-2 (Arabidopsis 4). -CoAS intron-1) (locus At3g21320, GenBank NC 003074). Both of the two homologous sequence-1 and homologous sequence-2 in the donor vector were identical to the corresponding homologous sequence-1 and homologous sequence-2 in the target vector (pDAB1585).

  To construct the donor vector, 299 bp of the 5 'partial PAT coding sequence was converted to Picoscript Ltd. , LLP, (Houston, Texas) and fused with full-length Arabidopsis 4-CoAS intron-1 (locus At3g21320, GenBank NC 003074). NcoI and XhoI sites were added to the 5 'and 3' ends of the fragment, respectively. This synthesized DNA fragment was then digested with NcoI / XhoI and inserted into pDAB1575 at the same site to replace the full length PAT gene coding sequence and its 3'UTR. The resulting construct was named pDAB1576 (FIG. 29).

  Next, pDAB1576 was digested with AgeI and the entire fragment containing the 5 'partial PAT expression cassette flanked by homologous sequence-1 and homologous sequence-2 was inserted into the binary base vector, pDAB2407, at the same site. The resulting construct was named pDAB1600 (FIG. 30), which was a binary version of the donor vector for plant cell retransformation.

Design and construction of zinc finger nuclease expression vector The zinc finger-Fok1 fusion protein gene was driven by the CsVMV promoter and 5′UTR (Verdaguer et al., 1996, Plant Molecular Biology 31: 1129-1139). A. The Tumefaciens open reading frame-24 (orf-24) 3 'untranslated region (UTR) (Gelvin et al., 1987, European Patent No. EP222493) was also included in the cassette.

To produce these vectors, PCR the _C 2 _{H 2} controls and _{C 3} H variants thereof IL-1-Fok1 and Scd27-Fok1 coding sequence set forth in Examples 1 to 4 from their initial design After amplification, BbsI or NcoI and SacI sites were added to the 5 ′ and 3 ′ ends of the PCR fragment, respectively, and cloned into pDAB3731 (FIG. 31) digested with NcoI-SacI. The resulting plasmids were named pDAB4322 (FIG. 32), pDAB4331 (FIG. 33), pDAB4332 (FIG. 34), pDAB4333 (FIG. 35), pDAB4334 (FIG. 36), pDAB4336 (FIG. 37) and pDAB4339 (FIG. 38). All of these vectors contained attL1 and attL2 sites flanking the ZFN expression cassette and were compatible with the Gateway ™ cloning system (Invitrogen, Carlsbad, California).

Two sets of binary vectors were constructed for the IL-1-FokI fusion protein. One contained the PAT selectable marker gene and the other did not contain the PAT selectable marker gene. For the SCd27-FokI fusion protein, only a binary type vector without the PAT selection marker gene was constructed. To create a binary vector containing the PAT selectable marker gene, the IL-1-FokI fusion protein expression cassette in pDAB4322, pDAB4331, pDAB4332, pDAB4333, pDAB4334 and pDAB4336 was obtained from LR Clonase ™ Enzyme Mix (Invitrogen, Carlsbad, California) And cloned into pDAB4321 (FIG. 39) through the LR recombination reaction. The resulting plasmids were named pDAB4323 (FIG. 40), pDAB4341 (FIG. 41), pDAB4342 (FIG. 42), pDAB4343 (FIG. 43), pDAB4344 (FIG. 44), and pDAB4346 (FIG. 45). To create a binary vector that does not contain the PAT selectable marker gene, the C ₂ H ₂ IL-1-FokI, C ₃ H IL-1-FokI and Scd27-FokI expression cassettes in pDAB4331, pDAB4336, and pDAB4339, respectively, It was cloned into pDAB4330 (FIG. 46) through an LR recombination reaction using (Trade Mark) Enzyme Mix (Invitrogen, Carlsbad, California). The resulting plasmids were named pDAB4351 (FIG. 47), pDAB4356 (FIG. 48) and pDAB4359 (FIG. 49), respectively.

To create a C ₂ H ₂ control for SCD27-Fok1, a HindIII / SacI fragment containing the CsVMV promoter and 5′UTR driving the PAT in pDAB7002 (FIG. 50) was excised from pDAB7025 (FIG. 51) with HindIII / SacI. In addition, the CsVMV promoter and 5′UTR and N. Substitution was made with a fragment containing the tabacam 5′UTR. The resulting plasmid was named pDAB1591 (FIG. 52). The Scd27-L0-FokI coding sequence was PCR amplified from their original vector pCDNA3.1-SCD27a-L0-FokI (FIG. 53) using primer pair P13 / P14. BbsI and SacI sites were added to the 5 ′ and 3 ′ ends of the PCR fragment, respectively. The PAT gene in pDAB1591 was replaced with a PCR fragment of the zinc finger fusion protein gene through SacI / NcoI cloning. The resulting plasmid was named pDAB1594 (FIG. 54). The binary form of this vector was constructed by excising a zinc finger fusion protein gene expression cassette from pDAB1594 as a PmeI / XhoI fragment, filling in the ends and cloning into pDAB2407 at the PmeI site. The resulting plasmid was named pDAB1598 (FIG. 55). Details of all binary vectors used for plant transformation are summarized in Table 6.

Design and production of positive control vector A vector containing a PAT gene expression cassette was used to estimate the frequency of illegitimate recombination and use it as a positive control. To be equivalent to the final recombinant, Arabidopsis 4-CoAS intron-1 (locus At3g21320, GenBank NC 003074) was replaced with 299/300 bp of the PAT coding sequence (Wohlleben et al., 1988, Gene 70: 25-37). Inserted into. To make this construct, the 2559 bp SwaI / ClaI fragment from pDAB1576 was ligated with the backbone fragment of pDAB1577 (FIG. 56) digested with the same restriction enzymes. The resulting vector is an Arabidopsis 4-CoAS intron-1 (locus At3g21320, GenBank NC 003074) (locus At3g21320, GenBank NC 003074) in the middle of the PAT coding sequence (Wohlleben et al., 1988, Gene 70: 25-37). PAT gene expression cassette with 1743 bp insertion). This vector was named pDAB1578 (FIG. 57).

  In order to produce a binary form of pDAB 1578, a PAT gene expression cassette having Arabidopsis thaliana intron-1 (locus At3g21320, GenBank NC 003074) was excised from pDAB1578 with PmeI / XhoI. The 3 'end of the fragment was blunt-ended and inserted into a binary basic vector, pDAB2407, at the PmeI site. It is driven by the Arabidopsis ubi10 promoter (Callis, et al., 1990, J. Biol. Chem. 265-12486-12493). Tumefaciens orf25 / 26 3'UTR (Gelvin et al., 1987, European Patent No. EP222493) PAT gene (Wohlleben et al. , 1988, Gene 70: 25-37), the resulting vector was named pDAB1601 (FIG. 58).

To confirm the C _{3 H} functional zinc finger nucleases in stimulating demonstrate intrachromosomal homologous recombination in the chromosome homologous recombination by re-transformation of a target cell culture in C 3 _H zinc finger nuclease gene, Two non-functional GFP fragments with a 540 bp overlap sequence were included in the target vector as shown in FIG. There was a GUS gene expression cassette between these two fragments. The IL-1-Fok1 fusion protein binding sequence was fused at its N-terminus with the GUS coding sequence. Without being bound by one theory, in the presence of the IL-1-Fok1 fusion protein, the IL-1 ZFN binding sequence is recognized and a double-stranded DNA break is induced, which is the endogenous DNA repair process. The hypothesis that it stimulates was made. In the absence of donor DNA, two partially homologous GFP fragments undergo an intrachromosomal homologous recombination process to reconstitute a functional GFP gene.

  A BY2-380 transgenic cell line containing a single full-length copy of the incorporated target sequence is placed in approximately 50-500 mg of callus tissue in 40-50 mL of LS basal medium containing 100 mg / L hygromycin, as described above. Used to resume suspension culture by subculture every 7 days. Prior to retransformation, suspension cultures were transferred to basal medium without hygromycin for at least two passages.

  Transformation of the target cell culture via Agrobacterium was performed as described above. For each experiment, 8 co-culture plates were made as follows. One plate contained cells co-cultured with 300 μL of basic Agrobacterium strain LBA4404; one plate contained cells co-cultured with 300 μL of Agrobacterium strain carrying pDAB1590 (functional GFP construct); Each of the 6 plates contained cells co-cultured with 300 μL of Agrobacterium strains carrying pDAB4323, pDAB4341, pDAB4342, pDAB4343, pDAB4344 and pDAB4346, respectively. After co-culture using the method described above, cells were plated on 8 plates containing LS basal medium supplemented with 500 mg / L carbenicillin without the selection reagent. Apparent expression of the constructed functional GFP gene produced visible fluorescence approximately 5-8 days after transformation. Count the number of green fluorescent loci per field by examining 8 “random” microscopic fields for each construct and 5 plates for each plate in each experiment and determine the mean value from 6 independent experiments. It was.

As summarized in Table 7, two C ₃ H zinc finger nucleases, pDAB4346 and pDAB4343, averaged 9.50 and 7.57 green fluorescent loci for each field, respectively. These two _{C 3} H design of IL-1-FokI have their _C 2 _{H 2} control, PDAB4341 (each field per 6.37 locus) and PDAB4323 (each field per 5.53 locus) better than It was a grade. On the other hand, compared to the C ₂ H ₂ control, the other two C ₃ H variants of the IL-1-FokI fusion protein, pDAB4344 (4.39 locus for each field) and pDAB4342 (0.3 for each field). The function of the 25 locus) was significantly impaired, especially in pDAB4342, where C ₃ H conversion was performed simply by replacing the second cysteine with histidine in the fourth finger. In a negative control transformed with the basic Agrobacterium strain, LBA4404, no appreciable fluorescence was observed beyond a slight background.

C 3 _H zinc finger nuclease gene and C _{3 H} zinc fingers in stimulating inter-chromosomal homologous recombination in demonstrating exemplary tobacco system inter-chromosomal homologous recombination by re-transformation of a target cell culture in the donor DNA sequence -Two strategies were developed and tested to confirm the functionality of the Fok1 fusion protein.

In strategy 1, the binding site for the zinc finger-Fok1 fusion protein (IL-1-L0-Fok1) was included in the middle of the target construct (FIG. 61). In this strategy, the binding sites are flanked by ˜3 kb non-homologous sequences on both sides, and upstream and downstream, respectively, homologous sequence-1 (N. tabacam RB7 MAR) and homologous sequence-2 (Arabidopsis 4-CoAS intron-1). Followed. As previously revealed (eg, US Patent Publication No. 20050064474), in the presence of a C ₂ H ₂ IL-1 zinc finger-Fok1 fusion protein, an IL-1-L0-Fok1 binding sequence is recognized, Double-stranded DNA breaks were induced at this specific site, which stimulated the endogenous DNA repair process. In the presence of donor DNA containing a homologous sequence identical to that in the target sequence, the 5 ′ partial PAT gene, along with its promoter, replaced the entire ˜6 kb DNA fragment between homologous sequences in the target through homologous recombination. . Through this process, the two partial PAT gene sequences, together with the intervening Arabidopsis 4-CoAS intron-1, reconstituted a functional PAT gene, resulting in a PAT expression and herbicide resistance phenotype.

  In strategy 2, two zinc finger-Fok1 binding sites (Scd27-L0-Fok1) were included in the target vector. One is N.I. Tabacam RB7 was just downstream of MAR, and the other was just upstream of Arabidopsis 4-CoAS intron-1 (FIG. 62). There was a ˜6 kb sequence between the two zinc finger-Fok1 fusion protein binding sites, which contained a 5 'GFP fragment, a GUS expression cassette and a 3' GFP fragment. As previously revealed (eg, US Patent Publication No. 20050064474), in the presence of the Scd27 zinc finger-Fok1 fusion protein, two binding sequences are recognized and double-stranded DNA breaks are induced at both positions. The ˜6 kb DNA fragment between these two binding sequences was removed, stimulating the endogenous DNA repair process. Similar to strategy 1, in the presence of donor DNA containing the same homologous sequence as that in the target sequence, the 5 ′ partial PAT gene, together with its promoter, is homologous recombination at the site where double-stranded DNA breakage is induced. Inserted into the target sequence. Through this process, the two partial PAT gene sequences, together with the intervening Arabidopsis 4-CoAS intron-1, reconstituted a functional PAT gene, resulting in a PAT expression and herbicide resistance phenotype.

Transformation of BY2-380 target cell culture via Agrobacterium was performed as described above. For each experiment, 12 co-culture plates were made as follows. One plate contained 50 μL of Agrobacterium strain carrying pDAB1600 (donor DNA) and 250 μL of Agrobacterium base strain LBA4404; one plate contained pDAB1601 (PAT selection marker). Cells containing 50 μL of bacterial strain and 250 μL of Agrobacterium base strain LBA4404; 2 plates contained 50 μL of Agrobacterium strain carrying pDAB1600 (donor DNA) and pDAB4351 (C ₂ H ₂ IL-1 ZFP) -Fok1) containing Agrobacterium strain 250μL co-cultured with cells bearing; three plates, pDAB1600 (Agrobacterium strain 50μL and pDAB4356 _(C 3 H harboring donor DNA) L-1 ZFP-Fok1) containing Agrobacterium strain 250μL co-cultured with cells bearing; two plates, Agrobacterium strain 50μL and pDAB1598 _{(C 2} H ₂ Scd carrying PDAB1600 (donor DNA) Cells were co-cultured with 250 μL of Agrobacterium strain carrying 27a ZFP-Fok1); three plates were 50 μL of Agrobacterium strain carrying pDAB1600 (donor DNA) and pDAB4359 (C ₃ H Scd27a ZFP-Fok1) Cells) co-cultured with 250 μL of Agrobacterium strain carrying After co-culture using the method described above, the cells were plated on LS basal medium containing 500 mg / L carbenicillin and 15 mg / L Bialabos®. Individual Bialaphos® resistant isolates appear 2-4 weeks after plating and are transferred to individual 60 × 20 mm plates (one isolate per plate) and are required for analysis. Until then, callus was maintained on a subculture schedule every 14 days.

A number of Bialaphos® resistant isolates were obtained from both C ₃ H IL-1 zinc finger nuclease (pDAB4356) and C ₃ H Scd27 zinc finger nuclease (pDAB4359). These isolates were analyzed by PCR using primer pair P24 / 25, which amplifies a DNA fragment spanning the entire rearranged PAT gene. Primer P24 was homologous to the 5 ′ end of the PAT coding sequence in the donor DNA and primer P25 was homologous to the 3 ′ end of the PAT coding sequence in the target DNA. When two partial PAT coding sequences are ligated through homologous recombination, a 2.3 kb PCR fragment results. As shown in FIG. 63, a 2.3 kb PCR product was obtained from a number of isolates analyzed. These isolates _were obtained from C 3 H IL-1 co-transformation of both zinc finger -Fok1 fusion protein gene / donor DNA and _C 3 H Scd27 zinc finger -Fok1 fusion protein gene / donor DNA. C ₃ H IL-1 zinc finger -Fok1 and _C 3 H Scd27 multiple independent 2.3 kb PCR product to agarose gel from isolates representative of those derived from both zinc finger -Fok1 fusion protein gene transformed And cloned into the pCR2.1 TOPO vector (Invitrogen, Carlsbad, California). The 2.3 kb PCR product was inserted into a TOPO vector and then sequenced using a Dye Terminator Cycle Sequencing Kit (Beckman Coulter). Sequencing results include recombinant sequences where all PCR products cloned into the TOPO vector contain the Arabidopsis 4-CoAS intron-1 intervening with the 5 ′ and 3 ′ partial PAT gene sequences as expected. It was confirmed. These results confirm the predicted chromosomal Hazama recombination strategies of both tested and illustrates the gene targeting by C 3 _H zinc finger -Fok1 expression of the fusion protein gene.

Identification of target gene sequences in maize cell cultures Sequence Identification In this example, the DNA sequence for an endogenous maize gene with a known function was selected as a target for genome editing using an engineered zinc finger nuclease. The genomic structure and sequence of this gene, termed IPP2-K, derived from the patent-protected maize inbred line 5XH751, is disclosed in WO 2006/029296, the disclosure of which is incorporated herein by reference. It is described in.

In particular, the IPP2-K genome sequence is used to query the TIGR corn genome database (available on the Internet at http://www.tigr.org/tdb/tgi/maize/) using the BLAST algorithm. used. Several additional genomic fragments were identified, including segments with overlapping homology to IPP2-K, including but not limited to accession numbers AZM515213 and TC311535. Based on the sequences of these accession numbers as well as the IPP2-K sequence, a number of short oligonucleotides can be obtained from the Primer 3 program (Rozen, S. and Skaletsky, HJ (2000) Primer 3 on the WWW for general users and for biologist programmers. In Designed for use as a PCR primer using: Krawetz S, Misener S (eds.) Bioinformatics Methods and Protocols: Methods in Molecular Biology Humana Press, Totowa, NJ, pp365-386; also available on the Internet) did. These primers include the following forward oligonucleotides:
1.5′-ATGGAGATGGATGGGGTTCTGCAAGCCGC-3 ′ (SEQ ID NO: 104)
2.5′-CTTGGCAAGGTACTGCGGCTCAAGAAGATTC-3 ′ (SEQ ID NO: 161)
3.5'-ATGAAGAAAGACAGGGGAATGAAGGAC-3 '(SEQ ID NO: 162)
4.5′-ATGAAGAAAGACAGGGGAATGAAGGACCGCCCAC-3 ′ (SEQ ID NO: 163)
5.5'-CATGGAGGGCGACGAGCGCGGTGTAGCTG-3 '(SEQ ID NO: 164)
6.5'-ATCGACCATGATTGGCACCCAGGGTTG-3 '(SEQ ID NO: 165)
Including, but not limited to.

In addition, the primer has the following reverse oligonucleotide:
7.5′-TTTCGACAAGCTCCAGAAAATCCCCTAGAAAC-3 ′ (SEQ ID NO: 166)
8.5'-ACAAGCTCCAGAAAAATCCCTAGAAACAC-3 '(SEQ ID NO: 167)
9.5'-TTCGACAAGCTCCAGAAAATCCCCTAGAAACAC-3 '(SEQ ID NO: 168)
10.5′-TGCTAAGAACAATTCTTTTCGACAAGCTCC-3 ′ (SEQ ID NO: 169)
11.5′-GAACATTCTTTTCGACAAGCTCCAGAAAACC-3 ′ (SEQ ID NO: 170)
Including, but not limited to.

  All oligonucleotide primers were synthesized by Integrated DNA Technologies (IDT, Coralville, IA) and purchased from the company.

B. Hi II corn cell culture To obtain immature embryos for initiating callus culture, F ₁ crosses between Hi-II parents A and B grown in a greenhouse (Armstrong, C., Green, C. and Phillips, R. (1991) Maize Genet. Coop. News Lett. 65: 92-93). Embryo about 1.0-1.2 mm in size (about 9-10 days after pollination) is taken from healthy ears and sterilized by scrubbing with Liqui-Nox® soap, Immersion in 70% ethanol for 2-3 minutes, then immersion in commercial 20% bleach (0.1% sodium hypochlorite) for 30 minutes.

The ears were washed with sterilized distilled water, and immature zygote embryos were aseptically excised and treated with 15 Ag10 medium (N6 medium (Chu CC, Wang CC, Sun CS, Hsu C., Yin KC, Chu CY, and Bi FY (1975 Sci. Sinica 18: 659-668), 1.0 mg / L 2,4-D, 20 g / L sucrose, 100 mg / L casein hydrolyzate (enzyme digest), 25 mM L-proline, 10 mg / L AgNO ₃ And 2.5 g / L Gelrite (pH 5.8), the scutellum was separated from the medium and cultured outward for 2-3 weeks. Tissues with the expected morphology (Welter, ME, Clayton, DS, Miller, MA, Petolino, JF. (1995) Plant Cell Rep: 14: 725-729) are selectively refreshed at twice weekly intervals Transfer to 15Ag10 medium for about 6 weeks, and then 4 weeks (N6 medium, 1.0 mg / L 2,4-D, 20 g / L sucrose, 100 mg / L casein hydrolyzate at intervals of twice a week for about 2 months) (Enzyme digest), 6 mM L-proline, 2.5 g / L gellite, pH 5.8).

  To initiate embryogenic suspension culture, about 3 ml of packed cell volume (PCV) of callus tissue originating from a single embryo was transferred to about 30 ml of H9CP + liquid medium (MS basal salt mixture (Murashige T., & Skoog F. (1962) Physiol. Plant. 15: 473-497) Modified MS vitamin containing 10 times lower nicotinic acid and 5 times higher thiamine-HCl, 2.0 mg / L 2,4-D, 2.0 mg / L α-naphthalene acetic acid (NAA), 30 g / L sucrose, 200 mg / L casein hydrolyzate (acid digest), 100 mg / L myo-inositol, 6 mM L-proline, 5% v / v coconut water (subculture) To pH 6.0). The suspension culture was maintained under dark conditions in a 125 ml Erlenmeyer flask on a temperature controlled shaker set at 125 rpm and 28 ° C. During cell line establishment (2-3 months), the suspension is passaged every 3.5 days by adding 3 ml of PCV and 7 ml of conditioned medium to 20 ml of fresh H9CP + liquid medium using a wide-mouth pipette. Subcultured. After reaching maturity, as evidenced by growth doubling, the suspension was scaled up and maintained in a 500 ml flask, thereby transferring 12 ml of PCV and 28 ml of conditioned medium to 80 ml of H9CP + medium. After complete establishment of the suspension culture, aliquots were stored frozen for later use. See International Publication No. WO2005 / 107437.

C. DNA Isolation and Amplification The corn HiII cell culture described above was placed in a standard GN6 medium (N6 medium, 2.0 mg / L 2,4-D, 30 g / L sucrose, 2.5 g / L gellite, pH 5 in a 250 ml flask. 8) and genomic DNA was extracted using the Qiagen (Valencia, CA) Plant DNeasy extraction kit according to the manufacturer's recommendations. PCR amplification reactions using the above primers in all possible combinations were performed under the following conditions. Reaction volume 25 μl containing 20 ng of gDNA template, 20 pmol of each primer, 1% DMSO and 10 units of Accuprime ™ Pf polymerase (Invitrogen, Carlsbad, Calif.) In the enzyme manufacturer's buffer. Amplification products having a size in the range of 500 bp-2 kb are 95 ° C.-1 min (95 ° C.-30 sec, 57-62 ° C.-30 sec, 72 ° C.-1 min) × 30, 72 ° C.-5 min. Resulted from an amplification cycle consisting of ° C-hold. The amplified fragment was cloned directly into the vector pCR2.1 (Invitrogen, Carlsbad, CA) using a TA cloning kit from Invitrogen (Carlsbad, CA) according to the manufacturer's recommendations.

D. Sequence analysis Previous analysis of the IPP2-K gene and HiII cell cultures in maize inbred line 5XH751 has indicated the presence of a few different genes, including a small gene family (Sun et al., In press, Plant Physiology; International Publication No. WO2006029296). Therefore, the isolated cloned fragment was sequenced with the CEQ Dye Terminator Cycle Sequencing Kit from Beckman Coulter (Fullerton, Calif.) According to the manufacturer's recommendations. Sequence analysis of a large number of clones revealed that two different gene fragments derived from two different, previously characterized loci of the corn genome were isolated from HiII cells.

  Comparison of two sequences isolated from HiII cultured cells shows that within the predicted coding region, minor differences, eg single nucleotide polymorphisms (SNPs), exist between the two paralogs, while introns and non-coding regions Indicated significantly different at the nucleotide level. These differences between the two paralogs are noted because they highlight regions of sequence that can be distinguished by sequence-dependent DNA binding proteins, such as zinc finger domains. One skilled in the art can design a zinc finger DNA binding domain that binds to one gene sequence and does not bind to another highly similar gene sequence. A 1.2 kb partial gene sequence corresponding to the subject paralog (FIG. 66) was selected as a template for zinc finger nuclease protein design and then subjected to the zinc finger DNA binding domain analysis described above.

Design of IPP2-K zinc finger DNA binding domain Using the target site identified for IPP2-K, a recognition helix for IPP2-K zinc finger was selected. The zinc finger design is shown in Table 8 below.

The target sites for zinc finger design are shown in Table 9 below.

  The IPP2-K design was incorporated into a zinc finger expression vector encoding a protein having a CCHC structure. See Tables 1-4 above. The non-canonical zinc finger coding sequence was then converted into the nuclease domain of type IIS restriction enzyme FokI (Wah et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10564-10569 with a 4-amino acid ZC linker. Of amino acids 384-579) to form IPP2-K ZFN.

Gene Repair Using IPP2-K Zinc Finger Nuclease The ability of IPP2-K ZFN described here to promote homologous recombination was demonstrated by Urnov (2005) Nature 435 (7042): 646-51 and US Patent Publication No. 20050064474 ( For example, it was tested in the GFP system described in Examples 6-11). Briefly, 50 ng of each ZFN and 500 ng of promoterless GFP donor (Urnov (2005) Nature) was applied to 500,000 reporter cells using 2 μL of Lipofectamine 2000 per sample according to the Invitrogen Lipofectamine 2000 protocol. Transfected.

  Vinblastine was added at a final concentration of 0.2 μM 24 hours after transfection and removed 72 hours after transfection.

  Cells were assayed for GFP expression by measuring 40,000 cells for each transfection on a Guava tabletop FACS analyzer 5 days after transfection. The results are shown in FIG.

Expression of C ₃ H ₁ ZFN in maize HiII cells Vector Design A plasmid vector for expression of ZFN protein in maize cells was constructed. To optimize the expression and relative stoichiometry of the two different proteins required to form a functional zinc finger nuclease heterodimer, both ZFNs can be combined into a single vector driven by a single promoter. An expression strategy that resulted in insertion of a multimeric open reading frame was employed. This strategy is based on the 2A sequence derived from Thesoa assigna virus (Mattion, NM, Harnish, EC, Crowley, JC & Reilly, PA (1996) J. Virol. 70, 8124-8127), opaque. ) -2 gene (op-2) maize nuclear localization signal (NLS) (Maddaloni, M., Di Fonzo, N., Hartings, H., Lazzaroni, N., Salaminil, F., Thompson, R) , & Motto M. (1989) Nucleic Acids Research Vol. 17 (18): 7532) and a promoter derived from maize ubiquitin-1 gene (Christensen AH, Sharrock RA, & Quail PH (1992) Plant Mol Biol. 18 ( 4) Use the functionality of 675-89). In order to develop these expression vectors for a given pair of ZFN-encoding genes selected from a library archive or newly synthesized, a step-wise modular cloning scheme was devised.

  First, a pVAX vector (see, eg, US Patent Publication No. 2005-0267061, the disclosure of which is incorporated herein by reference) contains an N-terminal expression domain as shown in FIG. 65, panels AE. Was modified as follows. This modified plasmid (pVAX-N2A-NLSop2-EGFP-FokMono) (FIG. 65A) is characterized by a redesigned and synthesized segment (RKRKESNRESARRSRYRK, SEQ ID NO: 133) encoding NLS derived from maize op-2. And a redesigned and synthesized segment encoding a FokI nuclease domain utilizing a corn codon bias. In addition, a single nucleotide insertion (C) downstream of the unique XhoI site created an extra SacI site for convenience of cloning.

  Second, the pVAX vector (see, eg, US Patent Publication No. 2005-0267061) was also modified to include a C-terminal expression domain. This modified plasmid (pVAX-C2A-NLSop2-EGFP-FokMono) (FIG. 65B) is characterized by a redesigned and synthesized segment (RKRKESNRERASARRSRYRK, SEQ ID NO: 133) encoding NLS derived from maize op-2. And a redesigned and synthesized segment encoding a FokI nuclease domain utilizing a corn codon bias. In addition, a 2A sequence (EGRGLSLTTCGD VEENGPP, SEQ ID NO: 134) from Tesoa Asinia virus was introduced at the N-terminus of the ZFN ORF for subsequent ligation of the two protein coding domains.

  Gene cassettes encoding ORFs of individual zinc finger proteins were cloned into either N2A or C2A vectors by ligation using restriction enzymes KpnI and BamHI to create compatible ends. Next, the BglII / XhoI fragment from the C2A vector was inserted into the N2A vector by the same restriction sites, generating an intermediate construct containing a cassette containing two ZFN coding domains flanked by NcoI and SacI restriction sites.

  Finally, the NcoI / SacI cassette from this intermediate construct (FIG. 65C) containing both ZFN genes was excised by restriction with these enzymes and ligated into the plasmid backbone pDAB3872 (FIG. 65D). The resulting plasmid contains the ZFN gene plus associated promoter and terminator sequences and a selectable marker for plasmid maintenance.

  In the final construct, an example of which is shown in FIG. 65E, the ZFN expression cassette (including promoter and terminator elements) is flanked by attL sites for convenient manipulation using the Gateway system from Invitrogen (Carlsbad, Calif.). . Each of the ZFN constructs created using this cloning scheme was transformed into E. coli DH5a cells (Invitrogen, Carlsbad, Calif.) And then maintained under appropriate selection.

B. DNA Delivery and Transient Expression Plasmid samples of the ZFN expression vector constructed as shown in FIG. 65E were prepared using antibiotic-free Gigaprep kit from Qiagen (Valencia, Calif.) According to the manufacturer's recommendations. Prepared from 2 L cultures of E. coli cells grown in LB medium. Plasmid DNA was delivered directly to corn HiII cultured cells using various methods.

  In one example, corn cells were subjected to DNA delivery by Whiskers ™ (Whisker). Approximately 24 hours prior to DNA delivery, PCV 3 ml of HiII corn suspension cells plus 7 ml of conditioned medium was subcultured into 20 ml of GN6 liquid medium (GN6 medium lacking gellite) in a 125 ml Erlenmeyer flask, 125 rpm, 28 Placed on a shaker at 0 ° C. for 24 hours. 2 mL of PCV was removed and 12 ml of GN6 S / M permeation medium in a 125 mL Erlenmeyer flask (N6 medium, 2.0 mg / L 2,4-D, 30 g / L sucrose, 45.5 g / L sorbitol, 45.5 g / L mannitol, 100 mg / L myo-inositol, pH 6.0). The flask was incubated in the dark for 30-35 minutes at 28 ° C. with gentle agitation (125 rpm). During this period, a 50 mg / ml suspension of Silicon Carbide Whiskers (Advanced Composite Materials, Inc., Eureka Springs, AK) is added by adding an appropriate volume of GN6 S / M liquid medium to a pre-weighed sterile whisker. Was prepared. After incubation in GN6 S / M, the contents of each flask were poured into a 15 mL conical centrifuge tube.

  After the cells settled, all but 1 mL of GN6 S / M liquid was removed and collected in a 125 mL flask for later use. The pre-wetted whisker suspension was vortexed at maximum speed for 60 seconds, 160 μL was added to the centrifuge tube using a wide-mouthed pipette tip, and 20 μg of DNA was added. The separator tube is “finger vortexed” and immediately placed in a Caulk “Vari-Mix II” dental amalgamator modified to hold a 17 × 100 mm culture tube at an intermediate speed of 60 Stir for 2 seconds. After agitation, the cell, medium, whisker and DNA cocktail was returned to the Erlenmeyer flask with an additional 18 ml of GN6 liquid medium. Cells were allowed to recover on a 125 RPM shaker for 2 hours at 28 ° C. in the dark.

  About 5-6 mL of the dispersed suspension was added to a Whatman No. using a glass cell collector unit connected to a house vacuum line so that 5-6 filters were obtained for each sample. It filtered with 4 filter papers (5.5cm). The filter was placed on a 60 × 20 mm plate of GN6 medium and cultured at 28 ° C. in the dark. After 24, 48 or 72 hours, cells from 2-5 filter papers were scraped, collected in tubes, placed on dry ice and then frozen at -80 ° C.

In another example of DNA delivery, purified endonuclease-free plasmid samples were delivered directly to corn cells using a micro-projectile bombardment adapted from the device manufacturer's protocol. All implants were performed on a Biolistic PDS-1000 / He ™ system (Bio-Rad Laboratories, Hercules, CA). For particle coating, 3 mg of 1.0 micron diameter gold particles were washed once with 100% ethanol and twice with sterile distilled water and resuspended in 50 μl of water in a siliconized Eppendorf tube. 5 μg of plasmid DNA, 20 μl of spermidine (0.1 M) and 50 μl of calcium chloride (2.5 M) were added to the gold suspension. The mixture was incubated at room temperature for 10 minutes, pelleted at 10K rpm for 10 seconds, resuspended in 60 μl cold 100% ethanol, and 8-9 μl was dispensed onto each macrocarrier. To prepare cells for implantation, a cell cluster was removed from the liquid culture on the third day after subculture, and a 2.5 cm diameter osmotic medium consisting of a Petri dish growth medium plus 0.256 M mannitol and sorbitol each was used. Placed in a circle. Cells were incubated in osmotic regulators for 4 hours prior to implantation. Implantation was performed in the apparatus described above according to the operating manual, placing the tissue on the middle shelf under conditions of 1100 psi and 27 inch Hg vacuum. At 24 hours after treatment, the implanted cell clusters were collected, frozen in liquid N ₂ and stored at −80 ° C.

Another example of ZFN DNA delivery and transient expression in maize cells involved the use of protoplast samples. HiII corn cell culture using a modified method from Mitchell and Petolino (1991) J. Plant. Physiol. 137: 530-536 and Lyznik et al. (1995) Plant J. 8 (2): 177-186. Protoplasts were prepared from Suspension cultures were harvested 48 hours after passage (mid-log phase) by centrifugation at 1000 rpm for 5 minutes. The culture medium was removed and 5 ml of loaded PCV was gently washed in 10 ml of W5 medium (154 mM NaCl ₂ ; 125 mM CaCl ₂ H ₂ O; 5 mM KCl ₂ ; 5 mM glucose; pH 5.8).

Washed cells were collected by centrifugation at 100 rpm for 5 minutes and then filter sterilized K3 medium (KNO ₃ 2.5 g; NH ₄ NO ₃ 250 mg; CaCl ₂ (dihydrate) 900 mg; Mg ₂ SO ₄ 250 mg _{; NH 4 SO 4 250mg; NaPO} 4 ( monobasic) 150 mg; xylose 250 mg; ferrous / Chealate stock (F 318) sulphate 10 ml; B5 micronutrients 1 ml (1000X stock - potassium iodide 750 mg; molybdate (sodium salt) Dehydrated 250 mg; Cobalt chloride 25 mg; Copper sulfate 25 mg); K3 vitamin 10 ml (100X stock solution-myo-inositol 1 g; Pyridoxine HCl 10 mg; Thiamine HCl 100 mg; Nicotinic acid 10 mg); +0.6 M mannitol; pH = 5.8] Incubated in an enzyme cocktail (Karlan Research Products Corp., Cottonwood, AZ) containing 3% cellulase Y-C + 0.3% pectinase Y23 in 5 ml, with gentle agitation to digest the secondary plant cell wall ( 50 rpm) and incubated at 25 ° C. for 5-6 hours.

After cell wall degradation, the enzyme-cell mixture was filtered through a 100 micron cell strainer and the flow-through containing protoplasts and cell debris was washed with an equal volume of K3 + 0.6M mannitol medium. Protoplasts were centrifuged at 800 rpm for 5 minutes, the supernatant was discarded, and washing was repeated. The protoplast pellet was washed and resuspended in 20 ml of K3 + 0.6M mannitol + 9% Ficoll 400 solution. Dispense 10 ml of this solution into two sterile plastic tubes and add 2 ml of TM medium (MES 19.52 g; mannitol 36.45 g; 40 ml of 2M CaCl ₂ · H ₂ O stock solution; pH = 5.5) onto the suspension. Gently overlapped to form a discontinuous gradient.

Viable protoplasts were separated from non-viable protoplasts, cell debris and intact suspension cells by centrifugation at 800 rpm for 5 minutes. A clear protoplast band formed at the gradient interface was pipetted off, washed with 10 ml of fresh TM solution and then centrifuged at 800 rpm for 5 minutes. The resulting protoplast pellet was resuspended in 1 ml TM medium and the number of viable protoplasts was quantified by 25 mg / mg fluorescein diacetate (FDA) staining on a hemocytometer. The protoplast solution was adjusted to a final concentration of 1 × 10 ⁷ protoplasts / ml in TM medium.

Approximately 1 × 10 ⁶ protoplasts (100 μl) were transferred to a 2 ml Eppendorf tube containing 10-80 μg of purified plasmid DNA. 100 μl of 40% PEG-3350 (Sigma Chemical Co., St. Louis, MO) solution was added dropwise and the suspension was gently mixed. The protoplast / DNA mixture was incubated at room temperature for 30 minutes and then diluted dropwise with 1 ml of GN6 growth medium. Diluted protoplasts were incubated in this medium for 24 hours at 25 ° C., then harvested, frozen in liquid N ₂ and stored at −80 ° C.

Functionality of ZFN in vivo The functionality of ZFN in this example is the ability of ZFN to be expressed in the cells of the crop species and the endogenous genome of the crop through its recognition of the desired target, binding to the desired target and its cleavage. It is understood that this includes (but is not limited to) the ability of ZFN to mediate double-strand breaks within. In the present example, it is also understood that the target of ZFN is an endogenous locus and a conformation gene in the crop genome.

  To assess whether the engineered ZFNs are functional for the predicted target gene in the genomic environment, a DNA sequence-based assay was performed. ZFN-induced double-stranded DNA breaks are repair mechanisms such as non-homologous end joining (NHEJ) (reviewed by Cahill et al., (2006) Mechanisms Front Biosci. 1 (11): 1958-76) Is expected to induce. One consequence of NHEJ is that a portion of the broken DNA strand is incompletely repaired, resulting in small deletions, insertions or substitutions at the cleavage site. One skilled in the art can detect these changes in the DNA sequence through a variety of methods.

A. PCR-Based Cloning and Sequencing In one example, maize HiII cultured cells expressing the ZFN protein are isolated 24 hours after transformation, frozen, and Qiagen (Valencia, CA) Plant DNeasy extraction kit is used according to manufacturer's recommendations The sample was subjected to genomic DNA extraction. PCR amplification was performed using oligonucleotide primers that are specific for the target gene of ZFN and flank the predicted cleavage site. A forward PCR primer specific for the target IPP2-K gene paralog (5′-GGAAGCATTTTCCAATTTGATGATAATGG-3 ′) (SEQ ID NO: 135) and reverse PCR primer (5′-CCCAAGGTCGGAGTTGTCCAATATGTTAC-3 ′) (SEQ ID NO: 136) This was used to amplify purified genomic DNA under the following conditions. A reaction volume of 25 μl containing 20 ng of gDNA template, 20 pmol of each primer, 1% DMSO and 10 units of Accuprime Pf polymerase (Invitrogen, Carlsbad, Calif.) In the buffer of the enzyme manufacturer. Amplification product of the expected size consists of 95 ° C.-1 min, (95 ° C.-30 sec, 61 ° C.-30 sec, 72 ° C.-1 min) × 30, 72 ° C.-5 min, 4 ° C.-hold. Resulted from the amplification cycle.

  The amplified fragment was cloned directly into the vector pCR2.1 (Invitrogen, Carlsbad, CA) using a TA cloning kit from Invitrogen (Carlsbad, CA). The isolated cloned fragment was sequenced in a 96-well format according to the manufacturer's recommendations with the CEQ Dye Terminator Cycle Sequencing Kit from Beckman Coulter (Fullerton, CA). In this experiment, the ZFN protein is predicted to bind to two short IPP2-K gene-specific sequences as shown in FIG. 66 to create a heterodimeric nuclease that cleaves double-stranded DNA.

Analysis of sequencing results from a number of clones was performed using clone no. Revealed that 127 contains a small deletion exactly at the predicted cleavage site of ZFN, indicating that the NHEJ mechanism was mediating incomplete repair of the DNA sequence at that site (FIG. 67).

  These results demonstrate the ability of these engineered ZFNs to specifically induce targeted double-strand breaks at endogenous loci within crop species.

B. Massively parallel sequencing analysis
In another example, a combination of PCR and massively parallel pyrosequencing was applied to query the genomes of multiple cell samples expressing different ZFN proteins targeted against this same sequence. Three variants of forward PCR primer (5′-XXXCACCCAAGTTGTATTGCCCTTCTCA-3 ′) (SEQ ID NO: 137) [where XXX = GGG, CCC or GGC] and reverse PCR primer (5′-XXXATAGGCTTGAGCCCAAGCAATCTT-3 ′) ( SEQ ID NO: 138) Three variants of [XXX = GCC, CCG or CGG] were synthesized (IDT, Coralville, IA). The 3 bp tag at the 5 'end of each primer serves as a key identifier, indicating which cell sample the amplicon is derived from. Primer pairs with matching identifier tags (keys) were used in combination to amplify purified genomic DNA from maize cell samples under the following conditions. 50 μl reaction volume containing 40 ng of gDNA template, 20 pmol of each primer, 1% DMSO and 10 units of Accuprime Pf polymerase (Invitrogen, Carlsbad, Calif.) In the enzyme manufacturer's buffer. Amplification products of the expected size resulted from amplification cycles consisting of 95 ° C-1 min (95 ° C-30 sec, 65 ° C-30 sec, 72 ° C-1 min) x 30, 4 ° C-hold, Was purified using the MinElute PCR purification kit from Qiagen (Valencia, CA) according to the manufacturer's recommendations.

  A massively parallel pyrosequencing reaction (also known as 454 sequencing) is described by 454 Life Sciences (Branford, CT) in (Margulies et al. (2005) Nature 437: 376-380). Performed directly on the product. Analysis of the 454 sequencing results was performed by identifying sequence reads that contained a deletion of the expected size and location within the DNA molecule.

  The results of these analyzes indicated the presence of numerous small deletions at the expected cleavage sites for these ZFNs, as shown in FIG. These deletions are located precisely at the ZFN target site, indicating that a double-strand break induced by ZFN occurred in the genome and was subsequently repaired by NHEJ. These results further demonstrate the ability of these engineered ZFNs to specifically induce targeted double-strand breaks at endogenous loci within crop species.

Designing Donor DNA for Targeted Integration In this example, it is understood that the donor DNA contains double stranded DNA molecules that are delivered to plant cells and integrated into the nuclear genome. The mechanism by which this integration occurs is homologous independent heterologous end joining (NHEJ; reviewed by Cahill et al., (2006) Mechanisms Front Biosci. 1: 1958-76) or two in nuclear DNA. This may be by another similar mechanism at the site of strand breaks. Such NHEJ-driven ligation-like integration into the genome of the donor DNA is referred to as random integration because the integration position of the donor DNA is mainly determined by the presence of double-stranded DNA breaks. In this mechanism, the integration of donor DNA into the genome does not depend on either the genomic nucleotide sequence of the cleavage site or the nucleotide sequence of the donor itself. Thus, in random integration, the “address” within the genome into which the donor DNA is integrated is not defined or predicted based on the sequence of the donor DNA. Random integration is the primary mechanism by which gene transfer of donor DNA occurs during standard plant transformation by DNA delivery to living plant cells via Agrobacterium or particle gun.

  For random integration, donor DNA can also be integrated into the genome by targeted integration. Targeted integration is reviewed in homology-dependent mechanisms such as homology-dependent single-strand annealing or homologous recombination (van den Bosch et al. (2002) Biol Chem. 383 (6): 873-892 It is understood that this occurs at the position (position) of double-strand break. In the case of homology-dependent DNA break repair, donor DNA comprising a nucleotide sequence having identity or similarity to the DNA at the break site can be incorporated at that site. Thus, the “address” at which the donor DNA is integrated into the genome depends on the nucleotide sequence identity or sequence similarity between the genome and the donor DNA molecule. In plant systems, double-strand break repair in DNA is known to utilize both NHEJ and homology-dependent pathways (reviewed in Puchta (2005) J. Exp. Bot. 56: 1-14). )

  In this example, we describe the design and construction of a donor DNA molecule that is integrated into the genome by targeted integration at the site of double-strand breaks induced by sequence-specific ZFN proteins. Various ZFN proteins can induce double-strand breaks at various nucleotides within the target gene sequence; the specific site of induced double-strand breaks is referred to as position.

  As described in Example 13, we characterized the nucleotide sequence of IPP2K from maize, the target gene. Subsequently, ZFN proteins that bind to specific bases of the target gene are designed (Example 14) and their binding / cleavage at that sequence in the target gene in heterologous systems and to endogenous genes in corn cells. The activity was verified (Examples 15 to 17). Here we describe the construction of various donor molecules designed for targeted integration into the maize genome at the location of ZFN-mediated double-strand breaks within the IPP2K gene. One skilled in the art would know to incorporate a ZFN-induced double-strand break by targeted integration driven by homology at any position in the genome where the nucleotide sequence is known and predicted to contain double-strand breaks. Designed donor DNA molecules can be constructed.

  In one embodiment described herein, the donor DNA molecule comprises an autonomous herbicide resistance gene expression cassette flanked by a segment of nucleotide sequence identical to the nucleotide sequence of the target gene, IPP2K at the target location. In this embodiment, it is understood that the autonomous herbicide tolerance cassette comprises a complete promoter-transcription unit (PTU) comprising a promoter, a herbicide tolerance gene, and a terminator sequence known to be functional in plant cells. Is done. One skilled in the art can select any combination of promoters, genes and terminators that constitute an autonomous PTU. Also included in this plasmid construct is a DNA fragment having sequence identity to the target gene (IPP2K) in maize at the indicated position. These fragments serve as “homology flanks” of the donor DNA and direct integration of this donor into the target gene at a specific location by targeted integration. The homology flank is located both upstream and downstream of the PTU in the correct 5'-3 'direction with respect to the PTU. One skilled in the art can envision homology flanks of various sizes and orientations in the construction of the donor DNA.

  In another embodiment described herein, the donor DNA molecule contains a plasmid construct comprising a non-autonomous herbicide resistance gene expression cassette flanked by a segment of nucleotide sequence identical to the nucleotide sequence of IPP2K at the target location. In this embodiment, it is understood that the non-autonomous herbicide tolerance cassette contains an incomplete promoter-transcription unit (PTU) lacking a functional promoter. Non-autonomous PTUs include herbicide resistance genes and terminator sequences known to be functional in plant cells. One skilled in the art can select any combination of genes and terminators that make up a non-autonomous PTU. In this example of a non-autonomous donor, the expression of the herbicide resistance gene relies on the integration of the donor segment at a genomic location in close proximity to a functional promoter that can drive the expression of that gene. A relatively rare situation can be envisaged where the donor is accidentally integrated into the locus where the promoter is present by random integration and can be used to drive the expression of the herbicide resistance gene. Alternatively, based on the presence of homologous flanks of DNA fragments of appropriate length having sequence identity to the target gene at a specific location in corn within the donor DNA construct, the exact target of the donor DNA to the target gene at the specific location Integration may occur (as described for autonomous donors) and thus the endogenous promoter of the target gene can be utilized. In this example, the homology flank is located both upstream and downstream of the PTU in the correct 5'-3 'direction relative to the PTU. One skilled in the art can envision homology flanks of various sizes and orientations in the construction of the donor DNA.

In both embodiments described herein (autonomous and non-autonomous donor designs), plasmid constructs typically include additional elements that allow cloning, expression of herbicide resistance genes, and subsequent analysis. . Such elements include bacterial replication origins, engineered restriction sites, etc. and are described below. One skilled in the art can envision the use of various elements including donor DNA molecules.
A. Bacterial strain and culture conditions

Escherichia coli strain (One Shot® Top 10 chemically competent cells; MAX Efficiency® DH5α ™ chemically competent cells, Invitrogen Life Technologies, Carlsbad, Calif., Luria Bertani Bros (B: / L bactotryptone, 10 g / L NaCl, 5 g / L Bacto yeast extract), LB agar (LB broth + 15 g / L bacto agar) or terrific broth (TB: 12 g / L bactotryptone, 24 g / L bacto yeast Extract, 0.4% v / v glycerol, 17 mM KH ₂ PO ₄ , 72 mM K ₂ HPO ₄ ) and grown at 37 ° C. for 16 hours. The liquid culture was shaken at 200 rpm. Chloramphenicol (50 μg / ml), kanamycin (50 μg / ml) or ampicillin (100 μg / ml) was added to the medium as needed. All antibiotics, culture media and buffer reagents used in this study were purchased from Sigma-Aldrich Corporation (St. Louis, MO) or Difco Laboratories (Detroit, MI).

B. Plasmid backbone position-1
A plasmid backbone containing a homology flank for IPP2K position-1 was engineered to allow integration of any donor DNA sequence into the corresponding target site of the IPP2K gene. One skilled in the art can envision a plasmid backbone that uses various cloning sites, modular design elements and sequences homologous to some target sequence in the genome of interest. The plasmid backbone exemplified here was derived from the basic plasmid vector pBC SK (−) phagemid (3.4 Kbp) (Stratagene, La Jolla, Calif.). The four-step synthesis described below was used to construct the position-1 plasmid backbone.

  Step No. In 1, a basic plasmid was prepared. 3 μg of pBCSK (−) was linearized at 37 ° C. for 1 hour using 10 units of SpeI restriction endonuclease and 10 units of NotI (New England Biolabs, Beverly, Mass.). Restriction DNA was added at 100 V in a 1.0% TAE (0.04 M Tris-acetate, 0.002 M EDTA) agarose gel supplemented with 0.5% ethidium bromide (Sigma-Aldrich Corporation, St. Louis, Mo.). Time electrophoresis was performed. DNA fragments were visualized with UV light and fragment size estimated by comparison with 1 Kbp DNA ladder (Invitrogen Life Technologies, Carlsbad, CA). The 3.4 Kbp SpeI / NotI digested subcloning vector, pBC SK (−), was gel excised and purified using the QIAquick Gel Extraction Kit (QIAGEN Inc., Valencia, Calif.) According to the manufacturer's instructions.

Step No. 2 isolated 5 ′ and 3 ′ homologous flank from position-1 of IPP2K. The following oligonucleotide primers are available from Integrated DNA Technologies, Inc. under standard desalting conditions. (Coralville, IA) and diluted with water to a concentration of 0.125 μg / μl.
5'-GCGGCCCGCGTCTCACCCGCGCTTGGGGATTGGATACGGAGCT-3 '(SEQ ID NO: 143)
5′-ACTAGTGATATGGGCCCCACAGGAGTTGCTCATGAACTTG-3 ′ (SEQ ID NO: 144)
5′-ACTTAGTCCAGAACTGGTTGAGTCGGTCAAACAGAATTTGCT-3 ′ (SEQ ID NO: 145)
5'-GTCGACCCTGATGCTACCATCATGGGCTGTTGT-3 '(SEQ ID NO: 146)

TaKaRa Biotechnology Inc. , Seta 3-4-1, Otsu, Shiga, 520-2193, Japan, a PCR amplification reaction was carried out using the following reagents. 10X LA PCR ^{_{(TM) Buffer II (Mg 2 +)}} 5μl, double-stranded gDNA template (corn HiII) 20 ng, positive oligonucleotide primer 10 pmol, reverse oligonucleotide primers 10 Pmol,dNTP mixture (each 2.5mM) 8μl, _{H 2} O 33.5 μl, TaKaRa LA Taq ™ DNA polymerase 0.5 μl (2.5 units), 1 drop of mineral oil. PCR reactions were performed using the Perkin-Elmer Cetus, 48 sample DNA Thermal Cycler (Norwalk, CT) under the following cycling conditions. 94 ° C., 4 minutes / cycle; 98 ° C., 20 seconds, 65 ° C., 1 minute, 68 ° C., 1 minute / 30 cycles; 72 ° C., 5 minutes / cycle; 4 ° C./hold. 15 μl of each PCR reaction was electrophoresed at 100 V for 1 hour in a 1.0% TAE agarose gel supplemented with 0.5% ethidium bromide. Amplified fragments were visualized with UV light and fragment size estimated by comparison with 1 Kbp DNA ladder. The expected amplification product was diagnosed by the presence of either 0.821 Kbp (5 ′ homology flank) or 0.821 Kbp (3 ′ homology flank) DNA fragment.

  These fragments were gel excised and purified using the QIAquick Gel Extraction Kit (QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions. The purified fragment was then transferred to TOPO TA Cloning® Kit (with pCR® 2.1 vector) and One Shot® Top 10 chemically competent E. coli cells (Invitrogen Life Technologies, Carlsbad, CA) was cloned into the pCR2.1 plasmid using the manufacturer's protocol.

  Individual colonies were inoculated into 14 ml falcon tubes (Becton-Dickinson, Franklin Lakes, NJ) containing 2 ml of TB supplemented with 50 μl / ml kanamycin and incubated for 16 hours at 37 ° C. with shaking at 200 rpm. Following incubation, 1.5 ml of cells were transferred to a 1.7 ml Costar microcentrifuge tube (Fisher Scientific, Pittsburgh, Pa.) And pelleted at 16,000 × g for 1 minute. The supernatant was removed and plasmid DNA was isolated as described above using the NucleoSpin® Plasmid Kit (BD Biosciences / Clontech / Machery-Nagel, Palo Alto, Calif.). 3 μg of the isolated plasmid from the 5 'homologous flank clone plasmid was digested with 10 units of SpeI and NotI each. Three prime-homology flank clone plasmids were digested with 10 units of SpeI and 20 units of SalI (New England Biolabs, Beverly, Mass.). All plasmid digests were incubated at 37 ° C. for 1 hour. Restriction DNA was electrophoresed at 100 V for 1 hour in a 1.0% TAE agarose gel supplemented with 0.5% ethidium bromide. Fragments were visualized with UV light and fragment size was estimated by comparison with a 1 Kbp DNA ladder. Expected plasmid clones include the presence of 0.821 Kbp (5 ′ homology flank) or 0.821 Kbp (3 ′ homology flank) insert DNA fragment in addition to the 3.9 Kbp pCR®2.1 vector Diagnosed by.

  Double-stranded sequencing reactions of plasmid clones were performed as described by the manufacturer using the CEQ ™ DTCS-Quick Start Kit (Beckman-Coulter, Palo Alto, Calif.). The reaction was purified using the Formata DTR Gel Filtration Cartridge (Edge BioSystems, Gaithersburg, MD) as described by the manufacturer's protocol. The sequence reaction was analyzed on a Beckman-Coulter CEQ ™ 2000 XL DNA Analysis System and nucleotide characterization was performed using Sequencer ™ version 4.1.4 (Gene Codes Corporation, Ann Arbor, MI). The sequence of the 0.821 Kbp fragment corresponding to the position-1 5'-homology flank derived from IPP2K is shown in Figure 87 (SEQ ID NO: 171). The sequence of the 0.821 Kbp fragment corresponding to the position-1 3'-homology flank derived from IPP2K is shown in Figure 88 (SEQ ID NO: 172).

  Step No. In 3, the position-1 5'-homology flank was ligated to the base plasmid. The restriction fragment corresponding to the clone containing the correct position-1 5 'homologous flank sequence was gel excised and purified using the QIAquick Gel Extraction Kit (QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions. The fragment corresponding to the position-1 5 ′ homology flank (0.821 Kbp) was then transferred to T4 DNA ligase (Invitrogen Life) in a reaction volume of 20 μl under conditions of 16 hours incubation in a 16 ° C. water bath (Technologies, Carlsbad, CA) was used to ligate the purified base plasmid digested with SpeI / NotI (Step No. 1) at a 1: 5 vector: insert ratio using 500 units. 5 μl of the ligation reaction was then transformed into E. coli One Shot® Top 10 chemically competent cells (Invitrogen Life Technologies, Carlsbad, Calif.) And plated under the selection conditions described by the manufacturer. . Individual colonies were inoculated into 14 ml falcon tubes (Becton-Dickinson, Franklin Lakes, NJ) containing 2 ml of TB supplemented with 50 μl / ml kanamycin and incubated for 16 hours at 37 ° C. with shaking at 200 rpm.

  Following incubation, 1.5 ml of cells were transferred to a 1.7 ml Costar microcentrifuge tube (Fisher Scientific, Pittsburgh, Pa.) And pelleted at 16,000 × g for 1 minute. The supernatant was removed and plasmid DNA was isolated as described above using the NucleoSpin® Plasmid Kit (BD Biosciences / Clontech / Machery-Nagel, Palo Alto, Calif.). 3 μg of the isolated plasmid DNA was digested with 10 units each of SpeI and NotI (New England Biolabs, Beverly, Mass.) And incubated at 37 ° C. for 1 hour. Restriction DNA was electrophoresed at 100 V for 1 hour in a 1.0% TAE agarose gel supplemented with 0.5% ethidium bromide. Fragments were visualized with UV light and fragment size was estimated by comparison with a 1 Kbp DNA ladder. The expected plasmid clone was diagnosed by the presence of the insert DNA fragment (5 'homology flank) of 0.821 Kbp in addition to the 3.4 Kbp basic plasmid.

  Step No. 4 the position-1 3 'homology flank Ligated to 3 products. Step No. 3 μg of the engineered product described in 3 was linearized at 37 ° C. for 1 hour with 10 units of SpeI restriction endonuclease and 20 units of SalI (New England Biolabs, Beverly, Mass.). Restriction DNA was added at 100 V in a 1.0% TAE (0.04 M Tris-acetate, 0.002 M EDTA) agarose gel supplemented with 0.5% ethidium bromide (Sigma-Aldrich Corporation, St. Louis, Mo.). Time electrophoresis was performed. DNA fragments were visualized with UV light and fragment size estimated by comparison with 1 Kbp DNA ladder (Invitrogen Life Technologies, Carlsbad, CA). ~ 4.2 Kbp SpeI / SalI digested step no. The product from 3 was gel excised and purified using the QIAquick Gel Extraction Kit (QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions.

  Step No. The isolated fragment of the 3'-homology flank donor (0.821 Kbp) prepared in step 2 was then digested with SpeI / SalI. Combined with 3 products, purified as described above as 20 μl of ligation reaction using a 1: 5 vector: insert ratio and 500 units of T4 DNA ligase (Invitrogen Life Technologies, Carlsbad, Calif.). The ligation reaction was incubated in a 16 ° C. water bath for 16 hours. After ligation, 5 μl of the ligation reaction was transformed into MAX Efficiency® DH5α ™ chemically competent cells (Invitrogen Life Technologies, Carlsbad, Calif.) According to manufacturer's recommendations. Individual colonies were inoculated into 14 ml falcon tubes (Becton-Dickinson, Franklin Lakes, NJ) containing 2 ml of TB supplemented with 50 μl / ml chloramphenicol.

  The culture was incubated for 16 hours at 37 ° C. with shaking at 200 rpm. Following incubation, 1.5 ml of cells were transferred to a 1.7 ml Costar microcentrifuge tube (Fisher Scientific, Pittsburgh, Pa.) And pelleted at 16,000 × g for 1 minute. The supernatant was removed and plasmid DNA was isolated as described above using the NucleoSpin® Plasmid Kit (BD Biosciences / Clontech / Machery-Nagel, Palo Alto, Calif.). 3 μg of the isolated plasmid was digested with 10 units each of SalI and NotI (New England Biolabs, Beverly, Mass.) And incubated at 37 ° C. for 1 hour. Restriction DNA was electrophoresed at 100 V for 1 hour in a 1.0% TAE agarose gel supplemented with 0.5% ethidium bromide. Fragments were visualized with UV light and fragment size was estimated by comparison with a 1 Kbp DNA ladder. The expected clone was diagnosed by the presence of two DNA fragments of 1.64 Kbp (insert) and 3.33 Kbp (base plasmid). The resulting plasmid was named pDAB7471 (FIG. 70).

C. Plasmid backbone position-2
The plasmid backbone containing the homology flank for IPP2K position-2 was engineered to allow integration of any donor DNA sequence into the corresponding target site of the IPP2K gene. One skilled in the art can envision a plasmid backbone that uses various cloning sites, modular design elements and sequences homologous to some target sequence in the genome of interest. The plasmid backbone exemplified here was derived from the basic plasmid vector pBC SK (−) phagemid (3.4 Kbp) (Stratagene, La Jolla, Calif.). The four-step synthesis described below was used to construct the position-2 plasmid backbone.

  Step No. In 1, a basic plasmid was prepared. 3 μg of pBCSK (−) was linearized at 37 ° C. for 1 hour using 10 units of SpeI restriction endonuclease and 10 units of NotI (New England Biolabs, Beverly, Mass.). Restriction DNA was added at 100 V in a 1.0% TAE (0.04 M Tris-acetate, 0.002 M EDTA) agarose gel supplemented with 0.5% ethidium bromide (Sigma-Aldrich Corporation, St. Louis, Mo.). Time electrophoresis was performed. DNA fragments were visualized with UV light and fragment size estimated by comparison with 1 Kbp DNA ladder (Invitrogen Life Technologies, Carlsbad, CA). The 3.4 Kbp SpeI / NotI digested subcloning vector, pBC SK (−), was gel excised and purified using the QIAquick Gel Extraction Kit (QIAGEN Inc., Valencia, Calif.) According to the manufacturer's instructions.

Step No. 2 isolated 5 ′ and 3 ′ homology flanks from position-2 of IPP2K. The following oligonucleotide primers are available from Integrated DNA Technologies, Inc. under standard desalting conditions. (Coralville, IA) and diluted with water to a concentration of 0.125 μg / μl.
5'-GCGGCCGCTAGATAGACAGATGCAGATGTC-3 '(SEQ ID NO: 147)
5′-ACTTAGTATGGCACCCAGGTGTTGGCTCA-3 ′ (SEQ ID NO: 148)
5'-ACTAGTCATGTCGATGGTGGGGTATGGTTCAGATTCAG-3 '(SEQ ID NO: 149)
5′-GTCGGACGTACAATGATTTCAGGTTACGGCCTCAGGAC-3 ′ (SEQ ID NO: 150)

TaKaRa Biotechnology Inc. , Seta 3-4-1, Otsu, Shiga, 520-2193, Japan, a PCR amplification reaction was carried out using the following reagents. 10X LA PCR ^{_{(TM) Buffer II (Mg 2 +)}} 5μl, double-stranded gDNA template (corn HiII) 20 ng, positive oligonucleotide primer 10 pmol, reverse oligonucleotide primers 10 Pmol,dNTP mixture (each 2.5mM) 8μl, _{H 2} O 33.5 μl, TaKaRa LA Taq ™ DNA polymerase 0.5 μl (2.5 units), 1 drop of mineral oil. PCR reactions were performed using the Perkin-Elmer Cetus, 48 sample DNA Thermal Cycler (Norwalk, CT) under the following cycling conditions. 94 ° C, 4 minutes / cycle; 98 ° C, 20 seconds, 55 ° C, 1 minute, 68 ° C, 1 minute / 30 cycles; 72 ° C, 5 minutes / cycle; 4 ° C / hold. 15 μl of each PCR reaction was electrophoresed at 100 V for 1 hour in a 1.0% TAE agarose gel supplemented with 0.5% ethidium bromide. Amplified fragments were visualized with UV light and fragment size estimated by comparison with 1 Kbp DNA ladder. The expected amplification product was diagnosed by the presence of either a DNA fragment of 0.855 Kbp (5 ′ homology flank) or 0.845 Kbp (3 ′ homology flank). These fragments were gel excised and purified using the QIAquick Gel Extraction Kit (QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions. The purified fragment was then transferred to TOPO TA Cloning® Kit (with pCR® 2.1 vector) and One Shot® Top 10 chemically competent E. coli cells (Invitrogen Life Technologies, Carlsbad, CA) was cloned into the pCR2.1 plasmid using the manufacturer's protocol.

  Individual colonies were inoculated into 14 ml falcon tubes (Becton-Dickinson, Franklin Lakes, NJ) containing 2 ml of TB supplemented with 50 μl / ml kanamycin and incubated for 16 hours at 37 ° C. with shaking at 200 rpm. Following incubation, 1.5 ml of cells were transferred to a 1.7 ml Costar microcentrifuge tube (Fisher Scientific, Pittsburgh, Pa.) And pelleted at 16,000 × g for 1 minute. The supernatant was removed and plasmid DNA was isolated as described above using the NucleoSpin® Plasmid Kit (BD Biosciences / Clontech / Machery-Nagel, Palo Alto, Calif.). 3 μg of the isolated plasmid from the 5 'homologous flank clone plasmid was digested with 10 units of SpeI and NotI each. Three prime-homologous flank clone plasmids were digested with 10 units of SpeI and 20 units of SalI (New England Biolabs, Beverly, Mass.). All plasmid digests were incubated at 37 ° C. for 1 hour.

  Restriction DNA was electrophoresed at 100 V for 1 hour in a 1.0% TAE agarose gel supplemented with 0.5% ethidium bromide. Fragments were visualized with UV light and fragment size was estimated by comparison with a 1 Kbp DNA ladder. Expected plasmid clones include the presence of 0.855 Kbp (5 ′ homology flank) or 0.845 Kbp (3 ′ homology flank) insert DNA fragment in addition to the 3.9 Kbp pCR®2.1 vector Diagnosed by.

  Double-stranded sequencing reactions of plasmid clones were performed as described by the manufacturer using the CEQ ™ DTCS-Quick Start Kit (Beckman-Coulter, Palo Alto, Calif.). The reaction was purified using the Formata DTR Gel Filtration Cartridge (Edge BioSystems, Gaithersburg, MD) as described by the manufacturer's protocol. The sequence reaction was analyzed on a Beckman-Coulter CEQ ™ 2000 XL DNA Analysis System and nucleotide characterization was performed using Sequencer ™ version 4.1.4 (Gene Codes Corporation, Ann Arbor, MI). The sequence of the 0.855 Kbp fragment corresponding to the position-2 5'-homology flank derived from IPP2K is shown in Figure 89 (SEQ ID NO: 139). The sequence of the 0.845 Kbp fragment corresponding to the position-2 3'-homology flank derived from IPP2K is shown in Figure 90 (SEQ ID NO: 140).

  Step No. In 3, the position-1 5'-homology flank was ligated to the base plasmid. A restriction fragment corresponding to a clone containing the correct position-2 5 'homologous flank sequence was gel excised and purified using the QIAquick Gel Extraction Kit (QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions. The fragment corresponding to the position-1 5 ′ homology flank (0.855 Kbp) was then added in a T4 DNA ligase (Invitrogen Life) in a reaction volume of 20 μl under conditions of 16 hours incubation in a 16 ° C. water bath. (Technologies, Carlsbad, CA) was used to ligate the purified base plasmid digested with SpeI / NotI (Step No. 1) at a 1: 5 vector: insert ratio using 500 units.

  5 μl of the ligation reaction was then transformed into E. coli One Shot® Top 10 chemically competent cells (Invitrogen Life Technologies, Carlsbad, Calif.) And plated under the selection conditions described by the manufacturer. . Individual colonies were inoculated into 14 ml falcon tubes (Becton-Dickinson, Franklin Lakes, NJ) containing 2 ml of TB supplemented with 50 μl / ml kanamycin and incubated for 16 hours at 37 ° C. with shaking at 200 rpm. Following incubation, 1.5 ml of cells were transferred to a 1.7 ml Costar microcentrifuge tube (Fisher Scientific, Pittsburgh, Pa.) And pelleted at 16,000 × g for 1 minute. The supernatant was removed and plasmid DNA was isolated as described above using the NucleoSpin® Plasmid Kit (BD Biosciences / Clontech / Machery-Nagel, Palo Alto, Calif.). 3 μg of the isolated plasmid DNA was digested with 10 units each of SpeI and NotI (New England Biolabs, Beverly, Mass.) And incubated at 37 ° C. for 1 hour. Restriction DNA was electrophoresed at 100 V for 1 hour in a 1.0% TAE agarose gel supplemented with 0.5% ethidium bromide. Fragments were visualized with UV light and fragment size was estimated by comparison with a 1 Kbp DNA ladder. The expected plasmid clone was diagnosed by the presence of a 0.855 Kbp insert DNA fragment (5 'homology flank) in addition to the 3.4 Kbp basic plasmid.

  Step No. 4 the position-2 3'-homology flank Ligated to 3 products. Step No. 3 μg of the engineered product described in 3 was linearized at 37 ° C. for 1 hour with 10 units of SpeI restriction endonuclease and 20 units of SalI (New England Biolabs, Beverly, Mass.). Restriction DNA was added at 100 V in a 1.0% TAE (0.04 M Tris-acetate, 0.002 M EDTA) agarose gel supplemented with 0.5% ethidium bromide (Sigma-Aldrich Corporation, St. Louis, Mo.). Time electrophoresis was performed. DNA fragments were visualized with UV light and fragment size estimated by comparison with 1 Kbp DNA ladder (Invitrogen Life Technologies, Carlsbad, CA). 4. 25 Kbp SpeI / SalI digested step no. The product from 3 was gel excised and purified using the QIAquick Gel Extraction Kit (QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions.

  Step No. The isolated fragment of the 3 'homologous flank donor (0.845 Kbp) prepared in step 2 was then digested with SpeI / SalI, step no. Combined with 3 products, purified as described above as 20 μl of ligation reaction using a 1: 5 vector: insert ratio and 500 units of T4 DNA ligase (Invitrogen Life Technologies, Carlsbad, Calif.). The ligation reaction was incubated in a 16 ° C. water bath for 16 hours. After ligation, 5 μl of the ligation reaction was transformed into MAX Efficiency® DH5α ™ chemically competent cells (Invitrogen Life Technologies, Carlsbad, Calif.) According to manufacturer's recommendations. Individual colonies were inoculated into 14 ml falcon tubes (Becton-Dickinson, Franklin Lakes, NJ) containing 2 ml of TB supplemented with 50 μl / ml chloramphenicol. The culture was incubated for 16 hours at 37 ° C. with shaking at 200 rpm. Following incubation, 1.5 ml of cells were transferred to a 1.7 ml Costar microcentrifuge tube (Fisher Scientific, Pittsburgh, Pa.) And pelleted at 16,000 × g for 1 minute. The supernatant was removed and plasmid DNA was isolated as described above using the NucleoSpin® Plasmid Kit (BD Biosciences / Clontech / Machery-Nagel, Palo Alto, Calif.). 3 μg of the isolated plasmid was digested with 10 units each of SalI and NotI (New England Biolabs, Beverly, Mass.) And incubated at 37 ° C. for 1 hour. Restriction DNA was electrophoresed at 100 V for 1 hour in a 1.0% TAE agarose gel supplemented with 0.5% ethidium bromide. Fragments were visualized with UV light and fragment size was estimated by comparison with a 1 Kbp DNA ladder. Expected clones were diagnosed by the presence of two DNA fragments of ~ 1.7 Kbp (insert) and 3.33 Kbp (base plasmid). The resulting plasmid was named pDAB7451 (FIG. 71).

D. Construction of an autonomous herbicide resistance gene expression cassette An autonomous herbicide resistance gene expression cassette containing a complete promoter-transcription unit (PTU) containing a promoter, herbicide resistance gene and a polyadenylation (polyA) termination sequence was constructed (FIG. 72). In this embodiment, the promoter sequence is derived from rice (O. sativa) actin 1 [McElroy et al. (Plant Cell 2, 163-171; 1990); GenBank accession S44221 and GenBank accession X63830]. To do. The herbicide resistance gene is a modified form of the PAT coding region derived from the herbicide bialaphos (originally Streptomyces viridochromogenes) (GenBank accession M22827; Wohlleben et al. Gene 70, 25- 37; 1988)) and contains a PAT (phosphinotricin acetyltransferase) gene. Modifications to the original sequence of the M22827 longest open reading frame are substantial and involve changing codon usage patterns to optimize expression in plants. Except for the substitution of valine as the first encoded amino acid by methionine and the addition of alanine as the second encoded amino acid, the protein encoded from the PAT open reading frame of pDAB3014 is the longest open of accession M22827. Identical to that encoded by the reading frame. Reconstructed PAT is found under GenBank accession I43995. The terminator sequence is corn (Z. mays) L. Lipase [L35913 corn lipase cDNA clone, except C at position 1093 of GenBank accession number L35913 is replaced by G at position 2468 in pDAB3014. This maize sequence is derived from the 3 ′ untranslated region / transcription terminator region for the PAT gene.

The following oligonucleotide primers are available from Integrated DNA Technologies, Inc. under standard desalting conditions. (Coralville, IA) and diluted with water to a concentration of 0.125 μg / μl.
5′-ACTTAGTTACTACTACTACTACTGAGGTCCATCATATGCTTGA-3 ′ (SEQ ID NO: 151)
5′-ACTAGTGGTGAATTCAGCACTTAAAGATCT-3 ′ (SEQ ID NO: 152)

TaKaRa Biotechnology Inc. , Seta 3-4-1, Otsu, Shiga, 520-2193, Japan, a PCR amplification reaction was carried out using the following reagents. 10X LA PCR ^{_{(TM) Buffer II (Mg 2 +)}} 5μl, double-stranded templates [pDAB3014 plasmid DNA] 20 ng, positive oligonucleotide primer 10 pmol, reverse oligonucleotide primers 10 Pmol,dNTP mixture (each 2.5mM) 8μl, _{H 2} O 33.5 μl, TaKaRa LA Taq ™ DNA polymerase 0.5 μl (2.5 units), 1 drop of mineral oil. PCR reactions were performed using the Perkin-Elmer Cetus, 48 sample DNA Thermal Cycler (Norwalk, CT) under the following cycling conditions. 94 ° C, 4 minutes / cycle; 98 ° C, 20 seconds, 55 ° C, 1 minute, 68 ° C, 3 minutes / 30 cycles; 72 ° C, 5 minutes / cycle; 4 ° C / hold. 15 μl of each PCR reaction was electrophoresed at 100 V for 1 hour in a 1.0% TAE agarose gel supplemented with 0.5% ethidium bromide.

  Amplified fragments were visualized with UV light and fragment size estimated by comparison with 1 Kbp DNA ladder. The expected amplification product was diagnosed by the presence of a 2.3 Kbp DNA fragment. This fragment was gel excised and purified using the QIAquick Gel Extraction Kit (QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions. The purified fragment is then cloned into the pCR2.1 plasmid using a TOPO TA Cloning® Kit, and One Shot® Top 10 chemically competent E. coli cells (Invitrogen Life Technologies) according to the manufacturer's protocol. , Carlsbad, CA).

  Individual colonies were inoculated into 14 ml falcon tubes (Becton-Dickinson, Franklin Lakes, NJ) containing 2 ml of TB supplemented with 50 μl / ml kanamycin and incubated for 16 hours at 37 ° C. with shaking at 200 rpm. Following incubation, 1.5 ml of cells were transferred to a 1.7 ml Costar microcentrifuge tube (Fisher Scientific, Pittsburgh, Pa.) And pelleted at 16,000 × g for 1 minute. The supernatant was removed and plasmid DNA was isolated as described above using the NucleoSpin® Plasmid Kit (BD Biosciences / Clontech / Machery-Nagel, Palo Alto, Calif.). 3 μg of the isolated plasmid was digested with 10 units of SpeI and NotI. All plasmid digests were incubated at 37 ° C. for 1 hour.

  Restriction DNA was electrophoresed at 100 V for 1 hour in a 1.0% TAE agarose gel supplemented with 0.5% ethidium bromide. Fragments were visualized with UV light and fragment size was estimated by comparison with a 1 Kbp DNA ladder. The expected plasmid clone was diagnosed by the presence of a 2.325 Kbp insert DNA fragment in addition to the 3.9 Kbp pCR®2.1 vector. Double-stranded sequencing reactions of plasmid clones were performed as described by the manufacturer using the CEQ ™ DTCS-Quick Start Kit (Beckman-Coulter, Palo Alto, Calif.). The reaction was purified using the Formata DTR Gel Filtration Cartridge (Edge BioSystems, Gaithersburg, MD) as described by the manufacturer's protocol. The sequence reaction was analyzed on a Beckman-Coulter CEQ ™ 2000 XL DNA Analysis System and nucleotide characterization was performed using Sequencer ™ version 4.1.4 (Gene Codes Corporation, Ann Arbor, MI).

E. Insertion of an autonomous herbicide resistance gene cassette into an autonomous donor of the plasmid backbone To create a donor plasmid, the autonomous herbicide resistance gene cassette described in Example 18D was incorporated into the plasmid backbone construct described in Examples 18B and 18C. Inserted. The restriction fragment derived from the clone containing the expected 2.325 Kbp sequence described above (FIG. 72) was gel excised and purified using the QIAquick Gel Extraction Kit (QIAGEN Inc., Valencia, Calif.) According to the manufacturer's instructions. .

  This fragment is then digested with the restriction enzyme SpeI and subsequently dephosphorylated with either purified pDAB7471 (position-1 plasmid backbone, FIG. 70) or pDAB7451 (position-2 plasmid backbone, FIG. 71). Combined in the ligation reaction. Ligation was performed under the following conditions. 1: 5 vector: insert ratio and 500 units of T4 DNA ligase (Invitrogen Life Technologies, Carlsbad, Calif.) In a reaction volume of 20 μl under conditions of 16 hours incubation in a 16 ° C. water bath. 5 μl of the ligation reaction was then transformed into 50 μl of E. coli MAX Efficiency® DH5α ™ chemically competent cells (Invitrogen Life Technologies, Carlsbad, Calif.) Under the selection conditions described by the manufacturer. Plated.

  Individual colonies were inoculated into 14 ml falcon tubes (Becton-Dickinson, Franklin Lakes, NJ) containing 2 ml of TB supplemented with 50 μl / ml chloramphenicol and incubated at 37 ° C. for 16 hours with shaking at 200 rpm. Following incubation, 1.5 ml of cells were transferred to a 1.7 ml Costar microcentrifuge tube (Fisher Scientific, Pittsburgh, Pa.) And pelleted at 16,000 × g for 1 minute. The supernatant was removed and plasmid DNA was isolated as described above using the NucleoSpin® Plasmid Kit (BD Biosciences / Clontech / Machery-Nagel, Palo Alto, Calif.). 3 μg of isolated plasmid DNA was digested with 10 units of SpeI (New England Biolabs, Beverly, Mass.) And incubated at 37 ° C. for 1 hour. Restriction DNA was electrophoresed at 100 V for 1 hour in a 1.0% TAE agarose gel supplemented with 0.5% ethidium bromide. Fragments were visualized with UV light and fragment size was estimated by comparison with a 1 Kbp DNA ladder. Expected plasmid clones were diagnosed by the presence of DNA fragments of 2.325 Kbp and ~ 4.9 Kbp (pDAB7471 vector) or 2.325 Kbp and ~ 5.0 Kbp (pDAB7451 vector).

  The resulting plasmids were named pDAB7422 (position-1 autonomous donor) (FIG. 73) and pDAB7452 (position-2 autonomous donor) (FIG. 74), respectively.

F. Construction of a non-autonomous herbicide resistance gene expression cassette A non-autonomous herbicide resistance gene expression cassette containing an incomplete promoter-transcription unit (PTU) was constructed (Figure 75). In this embodiment, the 2A sequence (Mattion, NM, Harnish, EC, Crowley, JC & Reilly, PA (1996) J. Virol. 70, 8124-8127) derived from Tesoa Asinia virus, herbicide resistance gene and A strategy that uses the functionality of the polyadenylation (polyA) termination sequence and does not use a promoter was used. In this embodiment, the 2A translation termination signal sequence was engineered to be translationally in frame with the herbicide resistance gene. In addition, the 2A / herbicide coding sequence was engineered to match the translational reading frame of the IPP2K gene target. The herbicide resistance gene is resistant to the herbicide bialaphos (originally a modified form of the PAT coding region derived from Streptomyces viridochromogenes (GenBank Accession M22827; Wohlleben et al. Gene 70, 25-37; 1988)). Containing the PAT (phosphinothricin acetyltransferase) gene. Modifications to the original sequence of the M22827 longest open reading frame are substantial and involve changing codon usage patterns to optimize expression in plants. Except for the substitution of valine as the first encoded amino acid by methionine and the addition of alanine as the second amino acid, the protein encoded from the PAT open reading frame of pDAB3014 is the longest open reading frame of M22827 (M22827 The same as that encoded by GTG at position 244). Reconstructed PAT is found under GenBank accession I43995. The terminator sequence is corn (Z. mays) L. Derived from the lipase [L35913 maize lipase cDNA clone, except that C at position 1093 of GenBank accession number L35913 is replaced by G at position 2468 in pDAB3014]. This maize sequence contains a 3 ′ untranslated region / transcription terminator region for the PAT gene.

The following oligonucleotide primers are available from Integrated DNA Technologies, Inc. under standard desalting conditions. (Coralville, IA) and diluted with water to a concentration of 0.125 μg / μl.
5'-ACTAGTGGCGGCGGGAGGGGCAGAGGAAGTCTCTCATACATGC
GGTGACGTGGAGGAAGACCCCGGCCCTAGGATGGCTTCTCCGGAGAGGAGAC
CAGTTGA-3 (SEQ ID NO: 153)
5′-ACTTAGTATGCATGTGAATTCAGCACTTAAAGATCT-3 ′ (SEQ ID NO: 154)

TaKaRa Biotechnology Inc. A PCR amplification reaction was carried out using the following reagents provided by (Seta 3-4-1, Otsu, Shiga, 520-2193, Japan). 10X LA PCR ^{_{(TM) Buffer II (Mg 2 +)}} 5μl, double-stranded templates [pDAB3014 plasmid DNA] 20 ng, positive oligonucleotide primer 10 pmol, reverse oligonucleotide primers 10 Pmol,dNTP mixture (each 2.5mM) 8μl, _{H 2} O 33.5 μl, TaKaRa LA Taq ™ DNA polymerase 0.5 μl (2.5 units), 1 drop of mineral oil. PCR reactions were performed using the Perkin-Elmer Cetus, 48 sample DNA Thermal Cycler (Norwalk, CT) under the following cycling conditions. 94 ° C., 4 minutes / cycle; 98 ° C., 20 seconds, 55 ° C., 1 minute, 68 ° C., 2 minutes / 30 cycles; 72 ° C., 5 minutes / cycle; 4 ° C./hold. 15 μl of each PCR reaction was electrophoresed at 100 V for 1 hour in a 1.0% TAE agarose gel supplemented with 0.5% ethidium bromide. Amplified fragments were visualized with UV light and fragment size estimated by comparison with 1 Kbp DNA ladder. The expected amplification product was diagnosed by the presence of a ˜1 Kbp DNA fragment. This fragment was gel excised and purified using the QIAquick Gel Extraction Kit (QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions. The purified fragment is then cloned into the pCR2.1 plasmid using a TOPO TA Cloning® Kit, and One Shot® Top 10 chemically competent E. coli cells (Invitrogen Life Technologies) according to the manufacturer's protocol. , Carlsbad, CA).

Individual colonies were inoculated into 14 ml falcon tubes (Becton-Dickinson, Franklin Lakes, NJ) containing 2 ml of TB supplemented with 50 μl / ml kanamycin and incubated for 16 hours at 37 ° C. with shaking at 200 rpm. Following incubation, 1.5 ml of cells were transferred to a 1.7 ml Costar microcentrifuge tube (Fisher Scientific, Pittsburgh, Pa.) And pelleted at 16,000 × g for 1 minute. The supernatant was removed and plasmid DNA was isolated as described above using the NucleoSpin® Plasmid Kit (BD Biosciences / Clontech / Macherey-Nagel, Palo Alto, Calif.). 3 μg of the isolated plasmid was digested with 10 units of SpeI. All plasmid digests were incubated at 37 ° C. for 1 hour. Restriction DNA was electrophoresed at 100 V for 1 hour in a 1.0% TAE agarose gel supplemented with 0.5% ethidium bromide. Fragments were visualized with UV light and fragment size was estimated by comparison with a 1 Kbp DNA ladder. Expected plasmid clones were diagnosed by the presence of insert DNA fragments of ˜1.0 Kbp and 3.9 Kbp (pCR®2.1 vector). Double-stranded sequencing reactions of plasmid clones were performed as described by the manufacturer using the CEQ ™ DTCS-Quick Start Kit (Beckman-Coulter, Palo Alto, Calif.). The reaction was purified using the Formata DTR Gel Filtration Cartridge (Edge BioSystems, Gaithersburg, MD) as described by the manufacturer's protocol. The sequence reaction was analyzed on a Beckman-Coulter CEQ ™ 2000 XL DNA Analysis System and nucleotide characterization was performed using Sequencer ™ version 4.1.4 (Gene Codes Corporation, Ann Arbor, MI).
G. Insertion of a non-autonomous herbicide resistance gene cassette into a non-autonomous donor of the plasmid backbone

  To create a donor plasmid, the non-autonomous herbicide resistance gene cassette described in Example 18F was inserted into the plasmid backbone construct described in Examples 18B and 18C. The restriction fragment corresponding to the clone containing the correct 1 Kbp sequence was gel excised and purified using the QIAquick Gel Extraction Kit (QIAGEN Inc., Valencia, Calif.) According to the manufacturer's instructions. This fragment was then digested with the restriction enzyme SpeI and subsequently dephosphorylated of purified pDAB7471 (position-1 plasmid backbone) (FIG. 70) or pDAB7451 (position-2 plasmid backbone) (FIG. 71). Either was combined in the ligation reaction. Ligation was performed under the following conditions. 1: 5 vector: insert ratio and 500 units of T4 DNA ligase (Invitrogen Life Technologies, Carlsbad, Calif.) In a reaction volume of 20 μl under conditions of 16 hours incubation in a 16 ° C. water bath. 5 μl of the ligation reaction was then transformed into 50 μl of E. coli MAX Efficiency® DH5α ™ chemically competent cells (Invitrogen Life Technologies, Carlsbad, Calif.) Under the selection conditions described by the manufacturer. Plated.

  Individual colonies were inoculated into 14 ml falcon tubes (Becton-Dickinson, Franklin Lakes, NJ) containing 2 ml of TB supplemented with 50 μl / ml chloramphenicol and incubated at 37 ° C. for 16 hours with shaking at 200 rpm. Following incubation, 1.5 ml of cells were transferred to a 1.7 ml Costar microcentrifuge tube (Fisher Scientific, Pittsburgh, Pa.) And pelleted at 16,000 × g for 1 minute. The supernatant was removed and plasmid DNA was isolated as described above using the NucleoSpin® Plasmid Kit (BD Biosciences / Clontech / Machery-Nagel, Palo Alto, Calif.). 3 μg of isolated plasmid DNA was digested with 10 units of SpeI (New England Biolabs, Beverly, Mass.) And incubated at 37 ° C. for 1 hour. Restriction DNA was electrophoresed at 100 V for 1 hour in a 1.0% TAE agarose gel supplemented with 0.5% ethidium bromide. Fragments were visualized with UV light and fragment size was estimated by comparison with a 1 Kbp DNA ladder. Expected plasmid clones were diagnosed by the presence of DNA fragments of 1.0 Kbp and 4.96 Kbp (pDAB7471 vector) or 1.0 Kbp and ˜5.0 Kbp (pDAB7451 vector). The resulting plasmids were named pDAB7423 (position-1 non-autonomous donor) (FIG. 76) and pDAB7454 (position-2 non-autonomous donor) (FIG. 77), respectively.

H. Position 1 ZFN + HR Donor Sequence: Combination Plasmid Selective to deliver two separate plasmids to one plant cell (eg, one plasmid contains the ZFN element and the second plasmid contains the herbicide resistant donor sequence) As a strategy, a single plasmid was engineered containing all the necessary elements described in this patent. The combination plasmids described in this example are targeted to specific IPP2K loci, where ZFN and autonomous PAT PTU and / or non-autonomous 2A / PAT PTU designed to cause double strand breaks and their Includes both donor flank designed to be incorporated into the tear site.

  To convert the vectors pDAB7422 and pDAB7423 (described in Examples 6E and 6G) into a Gateway® destination vector, site-specific recombination based on λ phage (Landy, A. (1989) Ann. Rev. Gateway (R) technology using Biochem. 55: 913) was utilized. Once converted, the plasmid containing the ZFN expression cassette (contained in the Gateway® entry vector) can be easily mobilized to the destination vector that creates the ZFN / donor combination plasmid. 1 μg of each such plasmid was digested with 10 units of NotI (New England Biolabs, Beverly, Mass.) For 1 hour at 37 ° C. The NotI restriction endonuclease was heat inactivated at 65 ° C for 15 minutes, after which the ends of the fragment were ring dephosphorylated at 37 ° C for 1 hour using 3 units of shrimp alkaline phosphatase (SAP) (Roche Diagnostics GmbH, Mannheim, Germany). did. Restriction DNA was electrophoresed at 100 V for 1 hour in a 1.0% TAE agarose gel supplemented with 0.5% ethidium bromide. Vector fragments (pDB7422 = 7.317 Kbp, pDAB7423 = 5.971 Kbp) were visualized with UV light, size estimated by comparison with a 1 Kbp DNA ladder, gel cut, and then QIAquick Gel Extraction Kit (QIAGEN Inc., QIAGEN Inc., The product was purified according to the manufacturer's instructions using Valencia, CA.

This vector fragment, then the ligation reaction was carried out under the following conditions, in combination Gateway (TM) Technology element, ATTrL, ccdB, and 2.274Kbp NotI fragment containing the Cm ^R and attR2. 1: 5 vector: insert ratio and 500 units of T4 DNA ligase (Invitrogen Life Technologies, Carlsbad, Calif.) In a reaction volume of 20 μl under conditions of 16 hours incubation in a 16 ° C. water bath. 5 μl of the ligation reaction was then transformed into 50 μl of E. coli One Shot® ccdB Survival ™ chemically competent cells (Invitrogen Life Technologies, Carlsbad, Calif.) Under the selection conditions described by the manufacturer And then plated.

  Individual colonies were inoculated into 14 ml falcon tubes (Becton-Dickinson, Franklin Lakes, NJ) containing 2 ml of TB supplemented with 50 μl / ml chloramphenicol and incubated at 37 ° C. for 16 hours with shaking at 200 rpm. Following incubation, 1.5 ml of cells were transferred to a 1.7 ml Costar microcentrifuge tube (Fisher Scientific, Pittsburgh, Pa.) And pelleted at 16,000 × g for 1 minute. The supernatant was removed and plasmid DNA was isolated as described above using the NucleoSpin® Plasmid Kit (BD Biosciences / Clontech / Machery-Nagel, Palo Alto, Calif.). 3 μg of isolated plasmid DNA was digested with 10 units of EcoRII (New England Biolabs, Inc., Beverly, Mass.) And incubated at 37 ° C. for 1 hour. Restriction DNA was electrophoresed at 100 V for 1 hour in a 1.0% TAE agarose gel supplemented with 0.5% ethidium bromide. Fragments were visualized with UV light and fragment size was estimated by comparison with a 1 Kbp DNA ladder. The expected plasmid clones were 1.448 Kbp for autonomous PAT PTU position-1 HR donors, 1.946 Kbp and 6.197 Kbp, and 5.807 Kbp and 2.438 Kbp for non-autonomous PAT position-1 HR donors. Diagnosed by the presence of DNA fragments. The resulting plasmids were named pDAB7424 (Gateway® compatible position-1 autonomous donor) (FIG. 78) and pDAB7425 (Gateway® compatible position-1 nonautonomous donor) (FIG. 79), respectively.

  As a result of these cloning operations, plasmids pDAB7424 and pDAB7425 were designated as Gateway® destination vectors. pDAB7412 has functionality as a Gateway (registered trademark) entry vector containing the following elements. ZwUbilv. 2 / ZFN12 / Zm Per5 3'UTR. LR Clonase ™ II (Invitrogen Life Technologies, Carlsbad, Calif.) To transfer a ZFN expression cassette (Gateway® entry vector) to an autonomous or non-autonomous donor molecule (Gateway® destination vector) Reactions were performed as outlined by the manufacturer at a ratio of 50 ng (entry vector); 150 ng / μl (destination vector). The resulting positive combination plasmids were named pDAB7426 (position-1 autonomous HR donor / ZFN12) (FIG. 80) and pDAB7427 (nonautonomous HR donor / ZFN12) (FIG. 81).

Delivery of ZFN and donor DNA into plant cells To enable integration of donor DNA into the plant genome via ZFN via targeted integration, followed by functional ZFNs in plant cells It is understood that protein expression is required. In addition, the functional ZFN protein induces double-strand breaks in the target DNA, and is then repaired by homologous driven integration of the donor DNA into the target locus so that the breakage of the donor DNA into the plant cell is restored. Delivery is also necessary. One skilled in the art can consider that expression of a functional ZFN protein can be achieved by a number of methods including, but not limited to, gene transfer of ZFN-encoding constructs or transient expression of ZFN-encoding constructs. In both of these cases, functional ZFN protein expression and donor DNA delivery in plant cells is achieved simultaneously to drive targeted integration.

  In the examples described herein, we demonstrate a method for simultaneous delivery of ZFN-encoding DNA and donor DNA to plant cells. One skilled in the art can use any of a variety of DNA delivery methods suitable for plant cells, including Agrobacterium-mediated transformation, particle gun-based DNA delivery or Whiskers ™. Including but not limited to mediated DNA delivery. In one embodiment described herein, whiskers ™ -mediated DNA delivery experiments were performed using various combinations of DNA constructs encoding ZFNs and donor DNA. These combinations include 1) a single plasmid containing both the ZFN coding sequence and donor DNA and 2) two separate plasmids, one containing the ZFN coding sequence and the other containing the donor DNA. In another embodiment, particle gun based DNA delivery was performed using various combinations of DNA constructs encoding ZFNs and donor DNA. Those skilled in the art can include these combinations 1) a single plasmid containing both the ZFN coding sequence and donor DNA and 2) two separate plasmids, one containing the ZFN coding sequence and the other containing the donor DNA. I can guess that.

A. DNA delivery via Whiskers ™ As described herein above, maize embryogenic Hi-II cell cultures were produced and used as a source of viable plant cells to reveal targeted integration. One skilled in the art may consider the use of cell cultures derived from various plant species, or differentiated plant tissue derived from various plant species, as a source of viable plant cells that reveal targeted integration.

  In this example, 12 ml of PCV from a cryopreserved cell line plus 28 ml of conditioned medium was added to GN6 liquid medium (N6 medium (Chu et al., 1975), 2.0 mg / L 2, in a 500 ml Erlenmeyer flask). 4-D, 30 g / L sucrose, pH 5.8) was subcultured to 80 ml and placed on a shaker at 28 ° C. and 125 rpm. This process was repeated twice using the same cell line so that a total of 36 ml of PCV was distributed across the three flasks. After 24 hours, the GN6 liquid medium was removed and GN6 S / M permeation medium (N6 medium, 2.0 mg / L 2,4-D, 30 g / L sucrose, 45.5 g / L sorbitol, 45.5 g / L mannitol, Exchanged with 72 ml of 100 mg / L myo-inositol, pH 6.0). The flask was incubated in the dark for 30-35 minutes at 28 ° C. with gentle agitation (125 rpm). During this incubation period, a 50 mg / ml suspension of silicon carbide whiskers (Advanced Composite Materials, LLC, Greer, SC) is prepared by adding 8.1 ml of GN6 S / M liquid medium to 405 mg of sterile silicon carbide whiskers. did.

  After incubation with GN6 S / M permeation medium, the contents of each flask were pooled in a 250 ml centrifuge bottle. After all the cells in the flask settled to the bottom, a volume of content greater than about 14 mL of GN6 S / M liquid was removed and collected in a sterile 1 L flask for later use. The pre-wetted whisker suspension was vortexed for 60 seconds at maximum speed and then added to the centrifuge bottle.

  In one example where a single plasmid containing both the ZFN coding sequence and donor DNA is delivered to plant cells, 170 μg of purified circular plasmid DNA was added to the jar. In the selective example of co-delivery of two separate plasmids, one containing the ZFN coding sequence and the other containing the donor DNA, a number of strategies regarding the amount of DNA were evaluated. One strategy used 85 μg donor DNA and 85 μg DNA encoding a zinc finger. Other modifications are based on the size of individual plasmids (kilobase pairs) so that a total of 170 μg of DNA is added per bottle, 10, 5 or 1-fold donor DNA versus 1-fold zinc. The molar ratio of finger DNA was used. In all cases of simultaneous delivery, the DNA was pre-pooled in tubes before being added to the centrifuge bottle. Once the DNA was added, the bottle was immediately placed in a modified Red Devil 5400 commercial paint mixer (Red Devequipment Co., Plymouth, Minn.) And stirred for 10 seconds. After stirring, a cocktail of cells, medium, whiskers and DNA was added to the contents of the 1 L flask along with 125 ml of fresh GN6 liquid medium to reduce osmoticant. Cells were allowed to recover for 2 hours on a shaker set at 125 rpm. Using a glass cell collector unit connected to a household vacuum cleaner, 6 mL of the dispersion suspension was obtained from a Whatman No. It filtered with 4 filter papers (5.5cm). The filter was placed on a 60 × 20 mm plate of GN6 solid medium (same as GN6 liquid medium except that 2.5 g / L gellite gelling agent was included) and cultured at 28 ° C. for 1 week in the dark.

B. DNA delivery via particle gun In the examples described herein, corn embryogenic suspensions were subcultured in GN6 liquid medium approximately 24 hours prior to the experiment as previously described herein. Excess liquid medium is removed and approximately 0.4 PCV cells are placed in a 2.5 cm diameter circle in the center of a 100 × 15 mm Petri dish containing GN6 S / M medium coagulated with 2.5 g / L gellite. Thinly spread. Cells were cultured for 4 hours under dark conditions. In order to coat the particle gun particles with DNA, 3 mg of 1.0 micron diameter gold particles were washed once with 100% ethanol and twice with sterilized distilled water, and resuspended in 50 μl of water in a siliconized Eppendorf tube. . 5 μg of plasmid DNA, 20 μl of spermidine (0.1 M) and 50 μl of calcium chloride (2.5 M) were separately added to the gold suspension and mixed by vortexing. The mixture was incubated at room temperature for 10 minutes, pelleted in a tabletop microcentrifuge at 10,000 rpm for 10 seconds, resuspended in 60 μl cold 100% ethanol, and 8-9 μl was dispensed to each macrocarrier.

  Implantation was performed using a Biolistic PDS-1000 / He ™ system (Bio-Rad Laboratories, Hercules, CA). The plate containing the cells was placed on the middle shelf under conditions of 1100 psi and 27 inch Hg vacuum and struck according to the operating manual. Sixteen hours after implantation, the tissue was transferred to GN6 (1H) medium as a small mass and cultured at 28 ° C. for 2-3 weeks under dark conditions. Metastasis continued every 2-4 weeks until the appearance of a putative transgenic isolate resulting from donor DNA integration. Identification, isolation and regeneration of putative donor DNA integration events caused by DNA delivery via particle gun are used for putative donor DNA integration events caused by DNA delivery via Whiskers ™ It is the same as the process described.

C. Identification and Isolation of Putative Target Integration Transgenic Events One week after DNA delivery, filter papers were washed with GN6 (1H) selective medium (N6 medium, 2.0 mg / L 2,4-D, 30 g / L sucrose, 100 mg. / L myo-inositol, 1.0 mg / L Bialaphos from Herbiace (Meiji Seika, Japan), 2.5 g / L gellite, pH 5.8) 60 × 20 mm plate. These selection plates were incubated for 1 week at 28 ° C. in the dark.

  After one week of selection in the dark, half of the cells from each plate were GN6 agarose medium maintained at 37-38 ° C (N6 medium, 2.0 mg / L 2 autoclaved at 121 ° C for 10 minutes). , 4-D, 30 g / L sucrose, 100 mg / L myo-inositol, 7 g / L SeaPlaque® agarose, pH 5.8) 3.0 mL and tissue by scooping into a tube containing 1 mg / L Vialaphos from Herbiace Were embedded in fresh medium.

  The agarose / tissue mixture was ground with a spatula and then 3 mL of the agarose / tissue mixture was evenly poured onto the surface of a 100 × 15 mm Petri dish containing GN6 (1H) medium. This process was repeated for both halves of each plate. Once all tissues were embedded, the plates were individually sealed with Nescofilm® or Parafilm M® and incubated at 28 ° C. for 10 weeks in the dark. Putative transformation isolates grown under these selection conditions were removed from the embedded plates and transferred to fresh selection medium in 60 × 20 mm plates. If sustained growth is evident after about 2 weeks, the event is considered resistant to the applied herbicide (bialaphos), and then an aliquot of cells is taken into a 2 mL Eppendorf tube for genotyping did.

  One of skill in the art can utilize a gene that encodes any suitable selectable marker in donor DNA and apply equivalent selection conditions to living cells. For example, an alternative selectable marker gene, such as AAD-1 described in WO 2005/107437 A2, can be used in the maize cells described herein as a donor for selection and recovery of integration events. .

Screening for Targeted Integration Events by PCR Genotyping Analysis In this example, PCR genotyping was performed by analyzing the polymerase chain of genomic DNA from isolated maize callus tissue that is predicted to contain donor DNA embedded in the genome. It is understood that this includes, but is not limited to, reaction (PCR) amplification, followed by standard cloning and sequence analysis of PCR amplification products. Methods for PCR genotyping have been widely described (eg, Rios, G. et al. (2002) Plant J. 32: 243-253) and genomes derived from any plant species or tissue type, including cell cultures. It can also be applied to DNA.

  Those skilled in the art include (but are not limited to) amplification of specific sequences within the plant genome, amplification of multiple specific sequences within the plant genome, amplification of non-specific sequences within the plant genome, or combinations thereof. ), Strategies for PCR genotyping can be devised. Amplification can be followed by cloning and sequencing, or direct sequence analysis of the amplified product, as described in this example. One skilled in the art may consider alternative methods for analysis of the amplification products generated herein.

  In one embodiment described herein, oligonucleotide primers specific for gene targets are used in PCR amplification. In another embodiment described herein, oligonucleotide primers specific for donor DNA sequences are used in PCR amplification. Another embodiment includes a combination of oligonucleotide primers that bind to both the gene target sequence and the donor DNA sequence. One skilled in the art can devise additional primer combinations and amplification reactions to perform genomic queries.

A. Genomic DNA extraction Genomic DNA (gDNA) was extracted from the isolated herbicide-tolerant maize cells described in Example 19 and used as a template for PCR genotyping experiments. About 100-300 μl packed cell volume (PCV) weeds isolated as described above according to the manufacturer's protocol detailed in gDNA, DNeasy® 96 Plant Kit (QIAGEN Inc., Valencia, Calif.) Extracted from drug resistant HiII callus. Genomic DNA was eluted in 100 μl elution buffer provided by the kit to a final concentration of 20-200 ng / μl and then analyzed by PCR-based genotyping as outlined below.

B. Primer design for PCR genotyping One skilled in the art can use various strategies for the design and implementation of PCR-based genotyping. Oligonucleotide primers designed to anneal to gene targets, donor DNA sequences and / or combinations of the two are feasible. Characterization of plasmid clones containing additional gene target sequence data by DNA sequencing to design oligonucleotide primers that can anneal to IPP2K gene targets in regions not included in the homology flank incorporated into the donor DNA molecule did. Double-stranded sequencing reactions of plasmid clones were performed as described by the manufacturer using the CEQ ™ DTCS-Quick Start Kit (Beckman-Coulter, Palo Alto, Calif.). The reaction was purified using the Formata DTR Gel Filtration Cartridge (Edge BioSystems, Gaithersburg, MD) as described by the manufacturer's protocol. The sequence reaction was analyzed on a Beckman-Coulter CEQ ™ 2000 XL DNA Analysis System and nucleotide characterization was performed using Sequencer ™ version 4.1.4 (Gene Codes Corporation, Ann Arbor, MI). These sequences correspond to regions of the IPP2K gene upstream (5 ′ side) and downstream (3 ′ side) of the ZFN target region, and are shown in FIG. 91 (SEQ ID NO: 141) and FIG. 92 (SEQ ID NO: 142). Yes.

In the examples presented here, all oligonucleotide primers were prepared under the conditions of standard desalting, Integrated DNA Technologies, Inc. (Coralville, IA) and diluted with water to a concentration of 100 μM. The following sets of forward and reverse oligonucleotide primers were designed to anneal to gDNA sequences specific for IPP2K gene targets located outside the boundaries of the donor DNA sequence. These oligonucleotides are as follows.
5'-TGGACGGAGCGAGAGCCAGAATTCGACGCTG-3 '(SEQ ID NO: 153)
5′-GTGCAAGAATGTATTGGGAATCAACCTGATG-3 ′ (SEQ ID NO: 154)

The second set of forward and reverse oligonucleotide primers also anneal to a gDNA sequence specific for the IPP2K gene target that is outside the boundaries of the donor DNA sequence but is still nested within the first pair. Designed.
5'-CGTTGGTACCAGTACTAGTACAGCATC-3 '(SEQ ID NO: 155)
5'-TCTTGGATCAAGGGCATCAAGCATTCCAATCT-3 '(SEQ ID NO: 156)

The forward and reverse oligonucleotide primers were additionally designed to specifically anneal to donor DNA corresponding to the coding region of the herbicide resistance gene.
5'-TGGGTAACTGGCCTAACTGG-3 '(SEQ ID NO: 157)
5′-TGGAAGGCTAGGAACGCCTTA-3 ′ (SEQ ID NO: 158)
5′-CCAGTTTAGCCAGTTACCCA-3 ′ (SEQ ID NO: 159)
5 'TAAGCGTTCCTAGCCTCTCCA-3' (SEQ ID NO: 160)

C. Donor DNA Specific PCR Amplification The primary PCR amplification reaction was performed by TaKaRa Biotechnology Inc. , Seta 3-4-1, Otsu, Shiga, 520-2193, Japan. 10X Ex Taq PCR ™ Buffer 2.5 μl, double-stranded genomic DNA template 40-200 ng, forward oligonucleotide primer 10 μM, reverse oligonucleotide primer 10 μM, dNTP mixture (2.5 mM each) 2 μl, H ₂ O 16 μl, Ex Taq ™ DNA polymerase 0.5 μl (2.5 units). PCR reactions were performed using the Bio-Rad, 96-sample DNA Engine Tetrad 2, Peltier Thermal Cycler (Hercules, CA) under the following cycling conditions. 94 ° C., 3 minutes / cycle; 94 ° C., 30 seconds, 64 ° C., 30 seconds, 72 ° C., 5 minutes / 35 cycles; 72 ° C., 10 minutes / cycle; 4 ° C./hold.

The amplification product of the primary PCR reaction was then reamplified in a secondary PCR reaction consisting of: 10 × Ex Taq PCR ™ Buffer 2.5 μl, template (1: 100 dilution of primary PCR reaction in H ₂ O) 2 μl, forward oligonucleotide primer 10 μM, reverse oligonucleotide primer 10 μM, dNTP mix (2.5 mM each) _{) 2μl, H 2 O 16μl,} Ex Taq ( TM) DNA polymerase 0.5 [mu] l (2.5 units). PCR reactions were performed using the Bio-Rad, 96-sample DNA Engine Tetrad 2, Peltier Thermal Cycler (Hercules, CA) under the following cycling conditions. 95 ° C, 1 minute / 1 cycle; 94 ° C, 15 seconds, 61 ° C, 30 seconds, 72 ° C, 30 seconds / 30 cycles; 72 ° C, 1 minute / 1 cycle; 4 ° C / hold. 10 μl of each amplification product was electrophoresed at 100 V for 1 hour in a 1.0% TAE agarose gel supplemented with 0.5% ethidium bromide. Amplified fragments were visualized with UV light and fragment size was estimated by comparison with 1 Kbp Plus DNA ladder (Invitrogen Life Technologies, Carlsbad, CA). PCR products containing the expected fragment were diagnosed by the presence of a 0.317 Kbp DNA fragment, as shown in FIG.

Detection of Target Integration Events For herbicide resistance events that include an integrated donor DNA molecule that encodes a herbicide resistance gene cassette, a fraction of the events are directed to the site of ZFN-induced double strand breaks. Expected to be a product of targeted integration of donor DNA. PCR-based genes using a combination of genome-specific PCR primers followed by genome-specific plus donor-specific PCR primers to distinguish these targeted integration events from those derived from random integration of herbicide resistance gene cassettes A type analysis strategy was used.

A. Genome-specific and then genome / donor-specific amplification In this embodiment, the primary PCR reaction used oligonucleotide primers specific for regions of the IPP2K gene target upstream and downstream of the donor integration region (eg, FIGS. 92 and 93). ). The primary PCR amplification reaction was performed using TaKaRa Biotechnology Inc. , Seta 3-4-1, Otsu, Shiga, 520-2193, Japan. 10X Ex Taq PCR ™ Buffer 2.5 μl, double-stranded corn gDNA template 40-200 ng, forward oligonucleotide primer 10 μM, reverse oligonucleotide primer 10 μM, dNTP mixture (2.5 mM each) 2 μl, H ₂ O 16 μl, Ex Taq ™ DNA polymerase 0.5 μl (2.5 units). PCR reactions were performed using the Bio-Rad, 96-sample DNA Engine Tetrad 2, Peltier Thermal Cycler (Hercules, CA) under the following cycling conditions. 94 ° C., 3 minutes / cycle; 94 ° C., 30 seconds, 64 ° C., 30 seconds, 72 ° C., 5 minutes / 35 cycles; 72 ° C., 10 minutes / cycle; 4 ° C./hold.

The primary PCR reaction product was then diluted 1: 100 in H ₂ O and used as template DNA for two different secondary PCR reactions. In this embodiment, the secondary reaction used primers and donor molecules that bind within the IPP2K genomic region, resulting in amplicons that span the integration boundary between the genome and donor. The initial reaction focused on the 5 'boundary between the genome and the donor. The second reaction focused on the 3 'boundary between the donor and the genome. Both reactions consisted of: 10 × Ex Taq PCR ™ Buffer 2.5 μl, template [1: 100 dilution of primary PCR reaction] 2 μl, forward oligonucleotide primer 10 μM, reverse oligonucleotide primer 10 μM, dNTP mixture (2.5 mM each) 2 μl, H ₂ O 16 μl, Ex Taq ™ DNA polymerase 0.5 μl (2.5 units). PCR reactions were performed using the Bio-Rad, 96-sample DNA Engine Tetrad 2, Peltier Thermal Cycler (Hercules, CA) under the following cycling conditions. 94 ° C., 3 minutes / cycle; 94 ° C., 30 seconds, 69 ° C., 30 seconds, 72 ° C., 2 minutes / 35 cycles; 72 ° C., 10 minutes / cycle; 4 ° C./hold. 20 μl of each secondary PCR reaction was electrophoresed at 100 V for 1 hour in a 1.0% TAE agarose gel supplemented with 0.5% ethidium bromide.

  Amplified fragments were visualized with UV light and fragment size was estimated by comparison with 1 Kbp Plus DNA ladder (Invitrogen Life Technologies, Carlsbad, CA). PCR products derived from the targeted integration of donors into the IPP2K gene were diagnosed by the presence of DNA fragments of 1.65 Kbp (5 'border) (Figure 83) or 1.99 Kbp (3' border) (Figure 84). These fragments were gel excised and purified using the QIAquick Gel Extraction Kit (QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions. The purified fragment was then transferred to TOPO TA Cloning® Kit (with pCR® 2.1 vector) and One Shot® Top 10 chemically competent E. coli cells (Invitrogen Life Technologies, Carlsbad, CA). ) Was cloned into the pCR2.1 plasmid using the manufacturer's protocol.

  Individual colonies were inoculated into 14 ml falcon tubes (Becton-Dickinson, Franklin Lakes, NJ) containing 2 ml of TB supplemented with 50 μl / ml kanamycin and incubated for 16 hours at 37 ° C. with shaking at 200 rpm. Following incubation, 1.5 ml of cells were transferred to a 1.7 ml Costar microcentrifuge tube (Fisher Scientific, Pittsburgh, Pa.) And pelleted at 16,000 × g for 1 minute. The supernatant was removed and plasmid DNA was isolated as described above using the NucleoSpin® Plasmid Kit (BD Biosciences / Clontech / Machery-Nagel, Palo Alto, Calif.). 3 μg of the isolated plasmid was digested with 10 units of Eco RI (New England Biolabs, Beverly, Mass.). All plasmid digests were incubated at 37 ° C. for 1 hour. Restriction DNA was electrophoresed at 100 V for 1 hour in a 1.0% TAE agarose gel supplemented with 0.5% ethidium bromide. Fragments were visualized with UV light and fragment size was estimated by comparison with a 1 Kbp Plus DNA ladder (Invitrogen Life Technologies, Carlsbad, Calif.).

  The expected plasmid clone was diagnosed by the presence of an appropriately sized insert DNA fragment in addition to the 3.9 Kbp pCR®2.1 vector. Double-stranded sequencing reactions of plasmid clones were performed as described by the manufacturer using the CEQ ™ DTCS-Quick Start Kit (Beckman-Coulter, Palo Alto, Calif.). The reaction was purified using the Formata DTR Gel Filtration Cartridge (Edge BioSystems, Gaithersburg, MD) as described by the manufacturer's protocol. The sequence reaction was analyzed on a Beckman-Coulter CEQ ™ 2000 XL DNA Analysis System and nucleotide characterization was performed using Sequencer ™ version 4.1.4 (Gene Codes Corporation, Ann Arbor, MI). Nucleotide alignment was performed using Vector NTi version 10.1 (Invitrogen Life Technologies, Carlsbad, CA).

  Analysis of sequence data from the target integration event (Event No. 073) was performed as follows. The primary PCR product over the entire integration site of the genome was subjected to secondary amplification focused on the 5 'or 3' boundary between the genome and the donor. The alignment of the wild-type IPP2K genomic sequence with the cloned fragments corresponding to these secondary amplification products, as well as the expected sequence of the target integration event, clearly indicated that correct integration of the donor DNA occurred at the target site.

  The nucleotide sequence of the IPP2K genomic locus, the genome / donor boundary, the nucleotide sequence of the donor region corresponding to the IPP2K homology flank and the nucleotide sequence of the herbicide resistance cassette are all in a number of cloned PCR products derived from this event. It was preserved. This event therefore represented a genome in which ZFN-mediated double-strand break homology-driven repair and targeted integration of donor DNA at specific gene targets occurred. Additional transformation events indicating the occurrence of unique target integration have been obtained, demonstrating that the method taught here is reproducible in corn callus. One skilled in the art can apply these methods to any genetic target of any plant species where targeted integration is desired.

B. Nested genome / donor specific amplification In this embodiment, both primary and subsequent secondary PCR reactions are combined with oligonucleotide primers specific for the donor sequence upstream of the donor integration region (appendices V and VI) or Oligonucleotide primers specific for the region of the downstream IPP2K gene target were used. In this example, the primary PCR amplification reaction was performed using TaKaRa Biotechnology Inc. , Seta 3-4-1, Otsu, Shiga, 520-2193, Japan. 10 × Ex Taq PCR ™ Buffer 2.5 μl, double-stranded maize gDNA template 40-200 ng, forward oligonucleotide 10 μM, reverse oligonucleotide primer 10 μM, dNTP mixture (2.5 mM each) 2 μl, H ₂ O 16 μl, Ex Taq TM DNA polymerase 0.5 μl (2.5 units). The PCR reaction was incubated under the following cycling conditions using a Bio-Rad, 96-sample DNA Engine Tetrad 2, Peltier Thermal Cycler (Hercules, CA). 94 ° C, 3 minutes / cycle; 94 ° C, 30 seconds, 52 ° C or 64 ° C, 30 seconds, 72 ° C, 2 minutes / 35 cycles; 72 ° C, 10 minutes / cycle; 4 ° C / hold.

The primary PCR reaction was then diluted 1: 100 in H ₂ O and used as template DNA for the secondary PCR reaction. In this embodiment, the secondary reaction also used primers and donor molecules that bind within the IPP2K genomic region, resulting in amplicons that span the integration boundary between the genome and donor. The specific primer used determines whether the amplification is focused on either the 5 ′ or 3 ′ boundary between the genome and the donor. The reagents for these reactions consisted of: 10 × Ex Taq PCR ™ Buffer 2.5 μl, template [1: 100 dilution of primary PCR reaction] 2 μl, forward oligonucleotide primer 10 μM, reverse oligonucleotide primer 10 μM, dNTP mixture (2.5 mM each) 2 μl, H ₂ O 16 μl, Ex Taq ™ DNA polymerase 0.5 μl (2.5 units). PCR reactions were performed using the Bio-Rad, 96-sample DNA Engine Tetrad 2, Peltier Thermal Cycler (Hercules, CA) under the following cycling conditions. 94 ° C, 3 minutes / cycle; 94 ° C, 30 seconds, 54 ° C or 60 ° C, 30 seconds, 72 ° C, 2 minutes / 35 cycles; 72 ° C, 10 minutes / cycle; 4 ° C / hold. 20 μl of each secondary PCR reaction was electrophoresed at 100 V for 1 hour in a 1.0% TAE agarose gel supplemented with 0.5% ethidium bromide.

  Amplified fragments were visualized with UV light and fragment size was estimated by comparison with 1 Kbp Plus DNA ladder (Invitrogen Life Technologies, Carlsbad, CA). PCR products derived from the targeted integration of donors into the IPP2K gene were diagnosed by the presence of DNA fragments at 1.35 Kbp (5 'boundary) (Figure 85) or 1.66 Kbp (3' boundary) (Figure 86). These fragments were gel excised and purified using the QIAquick Gel Extraction Kit (QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions. The purified fragment was then transferred to TOPO TA Cloning® Kit (with pCR® 2.1 vector) and One Shot® Top 10 chemically competent E. coli cells (Invitrogen Life Technologies, Carlsbad, CA). ) Was cloned into the pCR2.1 plasmid using the manufacturer's protocol.

C. Nucleotide sequence analysis of genotyping PCR products Individual colonies described in Example 21B were inoculated into 14 ml Falcon tubes (Becton-Dickinson, Franklin Lakes, NJ) containing 2 ml of TB supplemented with 50 μl / ml kanamycin at 200 rpm. Incubated for 16 hours at 37 ° C. with shaking. Following incubation, 1.5 ml of cells were transferred to a 1.7 ml Costar microcentrifuge tube (Fisher Scientific, Pittsburgh, Pa.) And pelleted at 16,000 × g for 1 minute. The supernatant was removed and plasmid DNA was isolated as described above using the NucleoSpin® Plasmid Kit (BD Biosciences / Clontech / Macherey-Nagel, Palo Alto, Calif.). 3 μg of the isolated plasmid was digested with 10 units of Eco RI (New England Biolabs, Beverly, Mass.). All plasmid digests were incubated at 37 ° C. for 1 hour. Restriction DNA was electrophoresed at 100 V for 1 hour in a 1.0% TAE agarose gel supplemented with 0.5% ethidium bromide. Fragments were visualized with UV light and fragment size was estimated by comparison with a 1 Kbp Plus DNA ladder (Invitrogen Life Technologies, Carlsbad, Calif.).

  Plasmid clones were diagnosed by the presence of the inserted DNA fragment in addition to the 3.9 Kbp pCR®2.1 vector. Double-stranded sequencing reactions of plasmid clones were performed as described by the manufacturer using the CEQ ™ DTCS-Quick Start Kit (Beckman-Coulter, Palo Alto, Calif.). The reaction was purified using the Formata DTR Gel Filtration Cartridge (Edge BioSystems, Gaithersburg, MD) as described by the manufacturer's protocol. The sequence reaction was analyzed on a Beckman-Coulter CEQ ™ 2000 XL DNA Analysis System and nucleotide characterization was performed using Sequencer ™ version 4.1.4 (Gene Codes Corporation, Ann Arbor, MI). Nucleotide alignment was performed using Vector NTi version 10.1 (Invitrogen Life Technologies, Carlsbad, CA).

  Sequence data including the boundary between upstream (5 ′) IPP2K genomic sequence and donor DNA from multiple targeted integration events is also included in the donor DNA and downstream (3 ′) IPP2K genome from multiple targeted integration events. Obtained, including sequence data including the boundaries between the sequences as well as sequence data including upstream (5 ′) boundary sequences derived from a single transformed callus event (No. 114). The transformation target integration event (No. 114) was the result of autonomous donor integration into the IPP2K gene target.

  In these analyses, both primary and secondary PCR amplification reactions focused on either the 5 'or 3' boundary between the genome and the donor. The alignment of the wild-type IPP2K genomic sequence with the cloned fragments corresponding to these secondary amplification products, as well as the expected sequence of the target integration event, revealed that donor DNA integration occurred at the target site. The nucleotide sequence of the IPP2K genomic locus, the genome / donor boundary, the nucleotide sequence of the donor region corresponding to the IPP2K homology flank and the nucleotide sequence of the herbicide resistance cassette are all in a number of cloned PCR products derived from this event. It was preserved.

  This event thus represents a genome in which homology-driven repair of double-strand breaks via ZFN at specific gene targets has occurred. Additional transformation events indicating the occurrence of unique target integration have been obtained, demonstrating that the method taught here is reproducible in corn callus. One skilled in the art can apply these methods to any genetic target of any plant species where targeted integration is desired.

Regeneration of Dwarf Intact Plants from Corn Callus Tissue Isolated callus of herbicide-resistant corn cells derived from HiII cell culture can be regenerated into intact dwarf corn plants. One skilled in the art can regenerate intact dwarf corn plants from various embryogenic corn cell cultures.

  In this example, isolated callus tissue was prepared from MS salt and vitamins, 30.0 g / L sucrose, 5 mg / L benzylaminopurine, 0.25 mg / L 2,4-D, 1 mg / L bialaphos, 2.5 g. Regeneration of isolated bialaphos-resistant HiII callus was initiated by transfer to / L gellite; cytokinin-based induction medium containing pH 5.7, 28 (1H). The cells were grown in low light (13 μEm-2s-1) for 1 week, then to a higher light (40 μEm-2s-1) condition and grown for another week. The cells were then transferred to a regeneration medium, 36 (1H), which was the same as the induction medium except that it lacked plant growth regulators. Small (3-5 cm) plants are cut out with hand tools and SHGA medium (Schenk and Hildebrandt basic salts and vitamins, 1972, Can. J. Bot 50: 199-204; 1 g / L myo-inositol, 10 g / L sucrose, 2.0 g / L gellite, pH 5.8) was placed in a sterile 150 × 25 mm glass culture tube.

  Once the plants have developed into sufficiently large differentiated roots and shoots, they are transplanted into 4 inch pots containing Metro-Mix 360 growth medium (Sun Gro Cultural Culture Ltd.) and placed in a greenhouse. It was. Cover the plant completely or partially with a clear plastic cup for 2-7 days, then transfer to a 5 gallon pot containing a mixture of 95% Metro-Mix 360 growth medium and 5% clay / loam soil until maturity Grow. Plants can be self-pollinated or cross-pollinated and cross-pollinated to produce T1 or F1 seeds, respectively. One skilled in the art can either self-pollinate the regenerated plant or cross-pollinate the regenerated plant with various germplasms to enable corn breeding.

  Additional information regarding targeted cleavage, targeted recombination, and targeted integration can be found in US Patent Application Publication No. US-2003-0232410, the disclosures of which are incorporated herein by reference in their entirety for all purposes; US-2005-0026157; US-2005-0064474; US-2005-0208489; and US-2006-0188987; and US patent applications filed July 26, 2006. 11 / 493,423.

  All patents, patent applications and patent publications referred to herein are hereby incorporated by reference in their entirety for all purposes.

  Although the disclosure has been provided in some detail by way of illustration and example for clarity of understanding, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit or scope of the disclosure. Accordingly, the foregoing description and examples should not be construed as limiting.

Claims (18)

A non-natural zinc finger protein comprising a non-canonical (non-C ₂ H ₂ ) zinc finger, wherein the non-canonical zinc finger has a helical portion involved in DNA binding, and at least one zinc finger has the sequence Cys − ^{_{(X A) 2-4 -Cys- (X}} B) 12 -His- (X C) 3-5 -Cys- (X D) 1-10 ( SEQ ID NO: 3) ^{wherein, X A,} X ^B, X ^C and X ^D may be any amino acid]
(I) the unnatural zinc finger protein has been engineered to bind to the target sequence;
(Ii) (X ^D ) _1-10 comprises an amino acid sequence selected from the group consisting of QKP, QLV and G residues at the C-terminus contacting the C-terminal Cys residue;
(Iii) When (X ^D ) _1-10 contains the amino acid sequence QKP and (X ^C ) consists of three amino acids, the residue of (X ^C ) is selected from the group consisting of IRT, IRR, TKI and AQR To be
Said non-natural zinc finger protein. The zinc finger protein according to claim 1, comprising any one of the sequences shown in SEQ ID NOs: 13-87 and 89-93 . X ^D is SEQ QLV, QKP, G, including GG or GGG, zinc finger protein of claim 1.   A zinc finger protein comprising a plurality of zinc fingers, wherein at least one of the zinc fingers comprises a CCHC zinc finger according to any one of claims 1 to 3. The zinc finger protein includes IPP2-K-1072a1, IPP2-K-1072b1, IPP2-K-1072c1, IPP2-K-r1065a1, IPP2-K-r1149a2, or IPP2-K-1156a2, and includes the IPP2-K gene The zinc finger protein according to any one of claims 1 to 4, wherein the zinc finger protein is engineered to bind to a target sequence.   A fusion protein comprising the zinc finger protein according to any one of claims 1 to 5 and one or more functional domains.   The fusion protein according to claim 6, wherein the functional domain comprises a cleavage half domain and the fusion protein further comprises a ZC linker interposed between the cleavage half domain and the zinc finger protein. The fusion protein according to claim 7, wherein the length of the ZC linker is 5 amino acids or 6 amino acids.   A polynucleotide encoding at least one zinc finger protein according to any one of claims 1 to 5 or at least one fusion protein according to any one of claims 6 to 8.   10. At least one zinc finger protein according to any of claims 1 to 5, at least one fusion protein according to any of claims 6 to 8, or at least one polynucleotide according to claim 9. Plant cells containing.   The plant cell according to claim 10, wherein the cell is a seed.   The plant cell according to claim 10 or 11, wherein IPP2-K is partially or completely inactivated, and the level of phytic acid in the seed is reduced. A method for targeted cleavage of cellular chromatin in plant cells comprising expressing in a plant cell a pair of fusion proteins according to claim 7 or at least one polynucleotide encoding a pair of said fusion proteins. There,
(A) the target sequence of the fusion protein is within 10 nucleotides of each other;
(B) the fusion protein dimerizes and cleaves the DNA located between the target sequences;
Said method. A method of targeted genetic recombination in a host plant cell, comprising:
(A) a host target locus wherein the pair of fusion proteins according to claim 7 or at least one polynucleotide encoding the pair of fusion proteins is expressed in a host plant cell, and a target sequence of the fusion protein is selected; Exist in the
(B) identifying a recombinant host plant cell that exhibits a sequence change at the host target locus, and (c) optionally introducing an exogenous polynucleotide into the host plant cell, wherein the exogenous polynucleotide is the host Including integration into the genome of a plant cell,
Said method.   15. The method of claim 14, wherein the sequence change is a mutation selected from the group consisting of genetic material deletion, genetic material insertion, genetic material replacement, and any combination thereof.   15. The method of claim 14, comprising reducing the level of phytic acid in the plant seed, making phosphorus in the plant seed more metabolically usable, inactivating or altering the IPP2-K gene. Method. A recombinant plant cell that is a progeny of any plant cell according to any one of claims 10-12. A plant cell produced by the method according to any one of claims 13 to 16.